A microscopic imaging device and method for liquid-based cell samples

Abstract

This invention relates to the field of microscopic imaging technology, and more particularly to a microscopic imaging device for liquid-based cell samples, comprising: a photosensitive area of ​​an image acquisition chip for acquiring image information of a liquid-based cell sample; at least one sample loading container disposed on the photosensitive area of ​​the image acquisition chip, with the lower surface of the sample loading container adhering to the upper surface of the photosensitive area of ​​the image acquisition chip, the lower surface of the sample loading container being transparent, and the sample loading container being used to load the liquid-based cell sample; each sample loading container comprising: a hollow cavity; two through holes symmetrically disposed on both sides of the sample loading container and respectively communicating with both ends of the hollow cavity, the two through holes being positioned above the lower surface of the sample loading container, and respectively used for inputting and outputting the liquid-based cell sample. The beneficial effect of this invention is that it allows the photosensitive area of ​​the image acquisition chip to be closer to the liquid-based cell sample, thereby improving the quality of the microscopic image.

Description

Technical Field

[0001] This invention relates to the field of microscopic imaging technology, and in particular to a microscopic imaging device and method for liquid-based cell samples. Background Technology

[0002] Microscopic imaging devices are used to optically magnify microbial tissues, observing and recording data through the photosensitive area of ​​an image acquisition chip to detect liquid-based cell samples and perform morphological analysis. Traditional optical microscopes are limited by the trade-off between field of view and resolution; that is, the higher the magnification of microbial tissues, the finer the images can be seen, but the field of view will also shrink.

[0003] In existing microscopic imaging devices, most employ a counting cell structure. For example, Chinese patent CN201520807546.8 discloses a microscope including a microscope platform and a counting cell platform assembly. The counting cell platform assembly has a counting cell and a fluid inlet tube connected to the counting cell. A frame is fixed to the microscope platform, and the counting cell platform assembly is located within the frame and held in place by the microscope platform. This patent adds a counting cell below the objective lens to the traditional optical microscope, replacing the traditional manual preparation of slides and smears. However, this method only solves the problems of complex slide preparation and unstable quality. It still relies on observation using a traditional optical microscope and cannot achieve full-field microscopic observation. Within a limited detection time, only a portion of the field of view can be selected for sampling observation and analysis, thus reducing the accuracy of detection and analysis. Furthermore, traditional optical microscopes are large and very expensive.

[0004] Furthermore, Chinese patent CN201811030915.1 discloses a bright-field and fluorescence dual-modal microscopic imaging system, which uses an image sensor chip to acquire images of the sample to be tested. The microscopic imaging system includes an image sensor chip and a sample chamber. The bottom of the sample chamber is not in direct contact with the image sensor chip; instead, a filter is placed between the image sensor chip and the sample chamber. During image acquisition, light refraction may occur, thus affecting the image acquisition quality.

[0005] Therefore, this invention proposes a novel microscopic imaging device and method for liquid-based cell samples. The photosensitive area of ​​the image acquisition chip is set in the recess of the carrier platform, and a sample loading container with a protrusion is designed. The protrusion is embedded in the recess of the carrier platform, so that the lower surface of the sample loading container is in contact with the photosensitive area of ​​the image acquisition chip, thereby improving the quality of the acquired image. Summary of the Invention

[0006] To address the aforementioned problems in the existing technology, a microscopic imaging device and method for liquid-based cell samples are provided.

[0007] The specific technical solution is as follows:

[0008] This invention provides a microscopic imaging device for liquid-based cell samples, comprising:

[0009] A photosensitive area of ​​an image acquisition chip is used to acquire image information of a liquid-based cell sample;

[0010] At least one sample loading container is disposed on the photosensitive area of ​​the image acquisition chip, and the lower surface of the sample loading container is attached to the upper surface of the photosensitive area of ​​the image acquisition chip. The lower surface of the sample loading container is transparent, and the sample loading container is used to load the liquid-based cell sample.

[0011] Each of the sample loading containers includes:

[0012] A hollow cavity;

[0013] Two through holes are symmetrically arranged on both sides of the sample loading container and are respectively connected to both ends of the hollow cavity. The two through holes are positioned above the lower surface of the sample loading container and are used for inputting and outputting the liquid-based cell sample, respectively.

[0014] Preferably, it includes a platform, the upper surface of which has a recess, and the photosensitive area of ​​the image acquisition chip is disposed in the recess.

[0015] Preferably, the sample loading container has a protrusion on the side facing the photosensitive area of ​​the image acquisition chip, the bottom of the protrusion being adapted to the size of the recess so that the protrusion is fitted into the recess, and the lower surface of the protrusion is in contact with the photosensitive area of ​​the image acquisition chip.

[0016] Preferably, the protrusion is inverted trapezoidal.

[0017] Preferably, the two through holes are respectively higher than the upper surface of the protrusion.

[0018] Preferably, the thickness of the hollow cavity is 50–200 μm.

[0019] Preferably, the sample loading container is entirely transparent.

[0020] Preferably, the apex angle of the protrusion is 90 to 180°.

[0021] Preferably, the thickness of the protrusion is not less than 200 μm.

[0022] Preferably, the hollow cavity is elliptical, circular, rectangular, or rounded parallelogram in shape.

[0023] Preferably, the sample loading container is made of transparent glass or a transparent organic polymer.

[0024] Preferably, the liquid-based cell sample is first labeled with a luciferase gene, then mixed with a luciferin substrate before being loaded into the sample loading container.

[0025] Preferably, an LED light source is provided above the microscopic imaging device.

[0026] Preferably, the microscopic imaging device further includes a light shield adapted to the LED light source, and the size of the light shield is adapted to the size of the sample loading container so that the light shield can cover the sample loading container.

[0027] Preferably, the detector array comprises a plurality of detector units, and the number of detector units is not less than 10 million.

[0028] Preferably, the size of each detector unit is no greater than 1 μm.

[0029] Preferably, the photosensitive area of ​​the image acquisition chip includes a plurality of pins, which are respectively led out from both sides of the recess.

[0030] Preferably, the microscopic imaging device further includes an image processor, wherein the photosensitive area of ​​the image acquisition chip is connected to the input terminal of the image processor through a plurality of pins, and the image processor is used to process the acquired image information.

[0031] Preferably, the microscopic imaging device further includes a display, the input of which is connected to the output of the image processor, for displaying the processed image information.

[0032] Preferably, the microscopic imaging device includes two sample loading containers, which are arranged side by side in the recess of the loading platform.

[0033] This invention also includes a liquid-based cell sample microscopic imaging method, applied to the aforementioned microscopic imaging device, specifically comprising the following steps:

[0034] Step S1: The liquid-based cell sample is diluted to form a diluent, and injected into the hollow cavity through one of the through holes of the sample loading container;

[0035] Step S2: Use the photosensitive area of ​​the image acquisition chip to acquire an image of the diluent in the hollow cavity to form the image information;

[0036] Step S3: The image processor performs optimization processing on the image information;

[0037] Step S4: Display the optimized image information on a monitor.

[0038] Preferably, step S1 further includes:

[0039] Step S11: Label the diluted solution using a luciferase gene;

[0040] Step S12: The labeled diluent is mixed with a fluorescein substrate.

[0041] Preferably, in step S2, before the image acquisition is performed, a light shield is used to seal the sample loading container so that the image acquisition is performed in a dark environment.

[0042] Preferably, the optimization process includes image enhancement processing and / or pseudo-coloring processing and / or image segmentation processing.

[0043] The present invention also includes a microfluidic chip for microscopic imaging of liquid-based cell samples, comprising a sample chamber for loading liquid-based cell samples and an imaging chip for image acquisition. The upper surface of the imaging chip is attached to the lower surface of one or more of the sample chambers. The sample chamber includes an inlet, an intermediate cavity, and an outlet. The inlet and outlet are respectively connected to the intermediate cavity. The lower part of the sample chamber has a trapezoidal boss structure, and the intermediate cavity is located on the trapezoidal boss.

[0044] The upper surface of the imaging chip is bonded to the lower surface of one or more of the sample chambers. This bonding means that the upper surface of the imaging chip and the lower surface of the sample chamber are joined without gaps, for example, by bonding with or without adhesive.

[0045] Alternatively, it can mean that the sample cavity and the imaging chip are integrally formed in terms of structure. Specifically, the above structure means that the sample cavity does not have a separate closed bottom plate structure, and the closed bottom plate used to accommodate the sample is implemented by the imaging chip.

[0046] The microfluidic chip can be composed of a single-channel sample chamber and an imaging chip bonded together, or it can be composed of multiple sample chambers and an imaging chip with a larger pixel scale bonded together to form a multi-channel chip.

[0047] Preferably, the detector array of the imaging chip is one of a semi-floating gate transistor array, a composite dielectric gate photodetector array, a dual-device photodetector array based on a composite dielectric gate, or a split-gate MOSFET imaging detector array.

[0048] The semi-floating gate transistor, for example, can be the semi-floating gate transistor structure described in the literature (Wang P, Lin X, Liu L, et al. A semi-floating gate transistor for low-voltage ultrafast memory and sensing operation.[J]. Science (New York, NY), 2013, 341(6146):640-643.), or the semi-floating gate transistor structure described in Chinese patent CN201410201614.6, including a semiconductor substrate (P-type); an N+ type source is formed in the semiconductor substrate by ion implantation, and a large N-type drain is formed by two-step ion implantation; a bottom dielectric, a semi-floating gate, a top dielectric, and a control gate are sequentially arranged on the semiconductor substrate, and a trench is formed in the middle of the bottom dielectric by etching, so that the semi-floating gate and the drain are in direct contact. Compared with the traditional floating gate transistor, which controls electron tunneling through the insulating dielectric layer by applying a high voltage, the semi-floating gate transistor adopts the quantum tunneling effect of silicon-based TFET and uses a PN junction diode to replace the traditional silicon oxide data erase window.

[0049] The composite dielectric gate photodetector, for example, can be the photosensitive composite dielectric gate MOSFET detector described in Chinese Patent CN200910024504.6. This photodetector includes: a semiconductor substrate (P-type); a bottom insulating dielectric, a photoelectron storage layer, a top insulating dielectric, and a control gate are sequentially disposed on the top of the semiconductor substrate; N-type source and drain electrodes are formed in the semiconductor substrate (near both sides of the stacked dielectric) through ion implantation doping. By applying a gate voltage greater than a threshold voltage to the control gate, the voltage difference between the source and drain is 0. A large voltage difference between the P-type substrate and the source terminal creates a relatively wide depletion region on the substrate. Cold electrons generated in the substrate are accelerated towards the gate under the influence of the electric field in the depletion region, gaining energy. When this energy exceeds the potential barrier between the substrate and the bottom dielectric, the electrons directly cross this barrier and enter the bottom dielectric. Under the influence of the electric field of the bottom dielectric, they move towards the gate at a very high speed, generating an injection current into the gate.

[0050] The dual-device photosensitive detection unit based on a composite dielectric grating can be, for example, the dual-device photosensitive detection unit based on a composite dielectric grating as described in Chinese Patent CN201610592997.3. The photosensitive detection unit includes a composite dielectric gate MOS-C portion with photosensitive function and a composite dielectric gate MOSFET portion with readout function, and these two portions are formed on the same P-type semiconductor substrate. The composite dielectric gate MOS-C portion includes a charge-coupled layer, a first top dielectric layer, and a first control gate sequentially stacked on the P-type semiconductor substrate, wherein an N-type injection layer is provided in the P-type semiconductor substrate below the charge-coupled layer. The composite dielectric gate MOSFET portion includes a bottom dielectric layer, the charge-coupled layer, a second top dielectric layer, and a second control gate sequentially stacked on the P-type semiconductor substrate, wherein an N-type source region and an N-type drain region are provided in the P-type semiconductor substrate on the side near the bottom dielectric layer, and a threshold adjustment injection region is provided in the P-type semiconductor substrate below the bottom dielectric layer. In the P-type semiconductor substrate, the N-type injection layer is separated from the N-type source region and the N-type drain region by a shallow trench isolation region and a P+ type injection region. During operation: a zero bias voltage is applied to the control gate, and a negative bias voltage pulse is applied to the substrate to form a depletion layer in the substrate. When light is incident on the depletion layer, photons are absorbed by the semiconductor, generating photoelectrons. Driven by the gate voltage, the photoelectrons move to the interface between the substrate and the gate oxide and accumulate at the interface, causing the threshold voltage of the read transistor to change, thereby characterizing the number of photoelectrons and converting the optical signal into a quantifiable electrical signal.

[0051] The split-gate MOSFET imaging detector, for example, can be the split-gate MOSFET imaging detector described in Chinese Patent CN201210349285.0. The structure of this imaging detector includes: two layers of insulating dielectric material and a control gate respectively disposed directly above a P-type semiconductor substrate, with a photoelectric storage layer disposed between the two insulating dielectric materials. At least one location on the control gate surface or substrate layer is a light-transparent or semi-transparent window within the detector's detection wavelength range. Select gates are disposed on both sides of the floating-gate MOSFET above the P-type semiconductor substrate, with an insulating dielectric layer between the select gates and the substrate. The insulating dielectric layer has the same material and thickness as the bottom insulating dielectric layer. An N-type semiconductor region is disposed on the P-type substrate surrounding the substrate controlled by the two select gates, forming the source and drain of the split-gate MOSFET. The two select gates are disposed on both sides of the floating-gate MOSFET, and the select gates are separated from the control gate and the photoelectric storage layer by an insulating dielectric material, which also separates the substrate controlled by the control gate from the source and drain of the imaging detector. The second insulating dielectric layer in contact with the control gate is a material that prevents the charge stored in the photoelectron storage layer from leaking to the control gate. The first insulating dielectric layer in contact with the substrate P-type semiconductor material, i.e., the bottom dielectric, effectively isolates the substrate channel under the control gate from the photoelectron storage layer. When the gate voltage is high enough or the incident photon energy is high, electrons in the channel are swept into the photoelectron storage layer. A positive bias pulse is applied to the control gate, a negative bias pulse is applied to the P-type semiconductor substrate, and a negative bias pulse is applied to both select gates. This forms a depletion layer in the P-type semiconductor substrate under the control gate. When light is incident on the depletion layer and photons are absorbed by the semiconductor, photoelectrons are generated. Driven by the gate voltage, the photoelectrons move to the interface between the channel and the bottom insulating layer. Because a negative bias is applied to both select gates, a high electron barrier is formed in the P-type semiconductor substrate controlled by the select gates. This high electron barrier effectively isolates the substrate controlled by the control gates from the N-type source and drain, ensuring that photoelectrons collected in the substrate depletion layer do not leak towards the source and drain. Simultaneously, electrons in the source and drain are also prevented from entering the substrate depletion layer by this high barrier. When the positive bias applied to the control gates is sufficiently large, photoelectrons collected in the substrate depletion layer will enter the photoelectron storage layer via FN tunneling. If the incident photon energy is high enough, exceeding the bandgap between the semiconductor and the underlying insulating dielectric layer, photoelectrons can enter the photoelectron storage layer via direct tunneling. During the photoelectron collection stage, the source and drain can be appropriately positively biased or left floating.

[0052] Preferably, the size of a single detector unit of the imaging chip is ≤1μm, and the detector array size is ≥10 million, in order to ensure a large field of view and high resolution when imaging liquid-based cell samples.

[0053] Preferably, the thickness of the intermediate cavity is between 50 and 200 μm.

[0054] Preferably, the thickness from the upper surface to the lower surface of the sample chamber is ≥1 mm.

[0055] Preferably, the angle of the apex of the trapezoidal boss is between 90° and 180°, and the thickness of the trapezoidal boss is ≥200μm.

[0056] Preferably, the shape of the intermediate cavity is elliptical, circular, rectangular, or rounded parallelogram.

[0057] Preferably, the sample chamber is completely transparent and is made of transparent glass or a transparent organic polymer.

[0058] Preferably, the liquid-based cell sample is selected from exfoliated cells in the urine, fecal dilution, pleural effusion, peritoneal fluid, cerebrospinal fluid, sputum, or tracheal mucus of mammals, or exfoliated cells from the oral cavity, gastric mucosa, or cervical scrapings of mammals, or blood cells or circulating tumor cells in the blood of mammals.

[0059] Preferably, the mammal refers to a human.

[0060] Preferably, the liquid-based cell sample is selected from exfoliated cells in the urine, fecal dilution, pleural effusion, peritoneal fluid, cerebrospinal fluid, sputum, or tracheal mucus of mammals, or exfoliated cells from the oral cavity, gastric mucosa, or cervical scrapings of mammals, or blood cells or circulating tumor cells in the blood of mammals after staining.

[0061] Preferably, the liquid-based cell sample is selected from exfoliated cells in the urine, fecal dilution, pleural effusion, peritoneal fluid, cerebrospinal fluid, sputum, or tracheal mucus of mammals, or exfoliated cells from the oral cavity, gastric mucosa, or cervical scrapings of mammals, or blood cells or circulating tumor cells in the blood of mammals that have been first labeled with the luciferase gene and then mixed with a luciferin substrate.

[0062] The present invention provides a liquid-based cell sample microscopic imaging method based on the above-mentioned device, comprising the following steps:

[0063] (1) Connect the imaging chip to an existing image processing module, wherein the image processing module is used to process the image data of the sample to be detected received by the imaging chip;

[0064] (2) Take the diluent of the liquid-based cell sample to be tested and inject it into the intermediate cavity through the inlet;

[0065] (3) Turn on the LED light source directly above the microscopic imaging device or cover it with a light shield. The imaging chip will start recording the projected microscopic image of the cell sample and obtain a full-field liquid-based cell sample microscopic image.

[0066] The purpose of covering the image with a light shield is to facilitate the imaging of autofluorescent samples, isolate the entire device from ambient light, and provide a dark environment for the imaging process. The light shield can be an opaque shell structure that needs to fit snugly with the microscopic imaging device below to form an opaque environment. The material of the light shield can be opaque plastic, opaque metal, or other opaque polymer materials, etc.

[0067] After receiving the autofluorescence image of the sample to be detected, the image processing module removes and suppresses noise in the autofluorescence image through relevant image processing algorithms, and performs image enhancement, pseudo-coloring and image segmentation on image data with poor imaging effect, such as image data with weak autofluorescence signal, blurred edges and low signal-to-noise ratio.

[0068] The image processing module can also be connected to an image analysis and display module, which is used to receive the processed image data for further interactive analysis and to display the final imaging result; the interactive analysis includes deleting, shrinking, enlarging, rotating, selecting specific regions, and labeling targets in the image.

[0069] The image processing module or image analysis and display module may also include an image storage module for establishing an image database, storing image data processed by the image processing module and analysis results and image data after interactive analysis by the image analysis and display module. The image storage module includes a database, which can be stored locally or on a cloud server. The image storage module saves the data processed by the image processing module and the analysis results or image data after processing by the image analysis and display module into the local database or uploads it to the cloud server via the network. It can also search, query, add, delete, modify, and back up the image data in the database.

[0070] The beneficial effects of the technical solution of this invention are as follows:

[0071] (1) Compared with traditional optical microscopes, the microscopic imaging device in this invention can replace the traditional method of manually making glass slides, and directly use the photosensitive area of ​​the image acquisition chip to record the microscopic imaging results of liquid-based cell samples, which simplifies the operation steps of detection and analysis, and can avoid inaccurate detection results due to insufficient experience of the detection personnel in making glass slides.

[0072] (2) Through the photosensitive area of ​​the image acquisition chip, high-resolution microscopic images of the entire field of view can be directly acquired without the need for magnification by an optical lens. Compared with the prior art of sampling microscopic observation by moving the microscope objective lens to extract several fields of view, the microscopic imaging in this invention has higher accuracy and stability.

[0073] (3) The photosensitive area of ​​the image acquisition chip is set in the recess of the carrier platform, and a sample loading container with a protrusion is designed. The protrusion is embedded in the recess of the carrier platform, so that the lower surface of the sample loading container is in contact with the photosensitive area of ​​the image acquisition chip, which shortens the distance between the photosensitive area of ​​the image acquisition chip and the liquid-based cell sample, making the photosensitive area of ​​the image acquisition chip closer to the liquid-based cell sample, thereby improving the quality of the microscopic image. Attached Figure Description

[0074] Embodiments of the invention will be described more fully with reference to the accompanying drawings. However, the drawings are for illustration and explanation only and do not constitute a limitation on the scope of the invention.

[0075] Figure 1 This is a schematic diagram of the overall structure of the microscopic imaging device in an embodiment of the present invention;

[0076] Figure 2 This is a schematic diagram of the sample loading container at the first observation angle in an embodiment of the present invention;

[0077] Figure 3 This is a schematic diagram of the sample loading container at the second observation angle in an embodiment of the present invention;

[0078] Figure 4 This is a side view of the sample loading container in an embodiment of the present invention;

[0079] Figure 5 This is a top view of the sample loading container in an embodiment of the present invention;

[0080] Figure 6 This is a schematic diagram of the detector array in the photosensitive area of ​​the image acquisition chip in an embodiment of the present invention.

[0081] Figure 7 This is a schematic diagram of the structure of a semi-floating gate transistor in an embodiment of the present invention;

[0082] Figure 8 This is a schematic diagram of the composite dielectric grating photodetector in an embodiment of the present invention;

[0083] Figure 9 This is the first type of structure of the dual-device photosensitive detection unit based on a composite dielectric grating in the embodiments of the present invention;

[0084] Figure 10This is the second type of structure of the dual-device photosensitive detection unit based on a composite dielectric grating in the embodiments of the present invention;

[0085] Figure 11 This is the third type of structure of the dual-device photosensitive detection unit based on a composite dielectric grating in the embodiments of the present invention;

[0086] Figure 12 This is the fourth type of structure of the dual-device photosensitive detection unit based on a composite dielectric grating in the embodiments of the present invention;

[0087] Figure 13 This is a schematic diagram of the structure of the split-gate MOSFET imaging detector in an embodiment of the present invention;

[0088] Figure 14 This is a schematic diagram of the structure of a microscopic imaging device with two sample loading containers in an embodiment of the present invention;

[0089] Figure 15 The image shows the microscopic imaging results of human blood sample dilution obtained using the microscopic imaging device and microscopic imaging method described in this embodiment of the invention.

[0090] Figure 16 This is a schematic diagram of the pin package structure of the photosensitive area of ​​an image acquisition chip in the prior art;

[0091] Figure 17 This is a schematic diagram of the pin package structure of the photosensitive area of ​​the image acquisition chip in an embodiment of the present invention;

[0092] Figure 18 This is a flowchart of the steps of the microscopic imaging method in the embodiments of the present invention;

[0093] Figure 19 This is a schematic diagram of the overall structure of the microfluidic chip in Embodiment 5 of the present invention;

[0094] Figure 20 This is a schematic diagram of the microfluidic chip in Embodiment 5 of the present invention from the first observation angle;

[0095] Figure 21 This is a schematic diagram of the microfluidic chip in Embodiment 5 of the present invention from a second observation angle;

[0096] Figure 22 This is a side view of the microfluidic chip in Embodiment 5 of the present invention;

[0097] Figure 23 This is a top view of the microfluidic chip in Embodiment 5 of the present invention;

[0098] Figure 24 This is a schematic diagram of the overall structure of the microfluidic chip in Embodiment Six of the present invention. Detailed Implementation

[0099] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0100] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0101] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0102] Example 1

[0103] The first embodiment of the present invention provides a microscopic imaging device for liquid-based cell samples, such as... Figure 1 As shown, it includes:

[0104] A photosensitive area 1 of an image acquisition chip is used to acquire image information of a liquid-based cell sample;

[0105] At least one sample loading container 2 is disposed on the photosensitive area 1 of the image acquisition chip, and the lower surface of the sample loading container 2 is attached to the upper surface of the photosensitive area 1 of the image acquisition chip. The lower surface of the sample loading container 2 is transparent. The sample loading container 2 is used to load liquid-based cell samples.

[0106] Each sample loading container 2 includes:

[0107] A hollow cavity 20;

[0108] Two through holes 21 are symmetrically arranged on both sides of the sample loading container 2 and are connected to both ends of the hollow cavity 20 respectively. The two through holes 21 are located above the lower surface of the sample loading container 2 and are used for inputting and outputting liquid-based cell samples respectively.

[0109] Specifically, in this embodiment, such as Figure 1As shown, the microscopic imaging device includes a photosensitive area 1 of an image acquisition chip and a sample loading container 2. The lower surface of the sample loading container 2 is completely attached to the upper surface of the photosensitive area 1 of the image acquisition chip, and the shape and size of the lower surface of the sample loading container 2 are adapted to the photosensitive area 1 of the image acquisition chip. The bottom of the sample loading container 2 is completely transparent, so that the liquid-based cell sample at the bottom of the sample loading container 2 can be completely exposed within the acquisition range of the photosensitive area 1 of the image acquisition chip, thereby realizing full-field microscopic observation and analysis. At the same time, since the lower surface of the sample loading container 2 is completely attached to the upper surface of the photosensitive area 1 of the image acquisition chip, the distance between the two is shortened, reducing the influence of light refraction on the imaging results and effectively improving the quality of the acquired image.

[0110] Specifically, the sample loading container 2 includes two through holes 21. The first through hole 21A on one side is used to inject liquid-based cell samples. After image acquisition and analysis of the liquid-based cell samples are completed, the liquid-based cell samples are output from the sample loading container 2 through the second through hole 21B on the other side. In addition, since the through holes 21 are arranged parallel to the photosensitive area 1 of the image acquisition chip, when cleaning the sample loading container 2, cleaning fluid can be introduced through one through hole and discharged through the other through hole, which is beneficial for the discharge of cleaning fluid.

[0111] As a preferred embodiment, such as Figure 1 As shown, the microscopic imaging device includes a platform 3, the upper surface of which has a recess 30, and the photosensitive area 1 of the image acquisition chip is disposed in the recess 30.

[0112] The sample loading container 2 has a protrusion 22 on the side facing the photosensitive area 1 of the image acquisition chip. The bottom of the protrusion 22 is adapted to the size of the recess 30 so that the protrusion 22 is snapped into the recess 30, and the lower surface of the protrusion 22 is in contact with the photosensitive area 1 of the image acquisition chip.

[0113] Specifically, in this embodiment, such as Figure 1 As shown, the upper surface of the platform 3 has a recess 30, and the photosensitive area 1 of the image acquisition chip is disposed in the recess 30. The shape and size of the recess 30 are adapted to the photosensitive area 1 of the image acquisition chip, so that the photosensitive area 1 of the image acquisition chip can fit snugly into the recess 30, which has a strengthening and fixing effect. Figure 5 As shown, the recess 30 is rectangular, and its periphery is used to encapsulate the pins of the photosensitive area 1 of the image acquisition chip.

[0114] It should be noted that the platform 3 is a single image acquisition chip. During the packaging process, encapsulating adhesive is used to fix the casing to the outer periphery of the image acquisition chip. This packaging method causes the outer periphery of the image acquisition chip to bulge, thus forming the recess 30. The photosensitive area of ​​the image acquisition chip is located in the recess 30, which causes the problem in the prior art where the bottom of the sample chamber cannot directly contact the photosensitive area.

[0115] Specifically, such as Figure 2 and 3 As shown, the sample loading container 2 in this embodiment has a protrusion 22, which is in the shape of an inverted trapezoid. The shape and size of the protrusion 22 are also adapted to the photosensitive area 1 of the image acquisition chip, so that the protrusion 22 is fitted into the recess 30, thereby making the lower surface of the protrusion 22 fit with the upper surface of the photosensitive area 1 of the image acquisition chip, thereby shortening the distance between the two and improving the imaging quality.

[0116] It should be noted that in this embodiment, the bottom of the protrusion 22 can be designed to be transparent, or the sample loading container 2 can be designed to be completely transparent. The transparent part can be made of transparent glass or transparent organic polymer. Preferably, it is made of K9 optical glass. K9 glass is a high-performance optical material, mainly used in optical coating and other fields. Its light transmittance, refractive effect, hardness and texture are far superior to resin materials.

[0117] In a preferred embodiment, the thickness of the sample loading container is not less than 1 mm, and in this embodiment, it is preferably 5 mm.

[0118] The thickness of the hollow cavity 20 is 50-200 μm, and is preferably 120 μm in this embodiment;

[0119] The thickness of the protrusion 22 is not less than 200 μm, and preferably 350 μm in this embodiment;

[0120] The angle of the apex of the protrusion 22 is between 90° and 180°, and is preferably 120° in this embodiment.

[0121] As a preferred embodiment, such as Figure 6 As shown, the photosensitive area 1 of the image acquisition chip includes a detector array composed of a plurality of detector units 10, the number of detector units 10 is not less than 10 million, and the size of each detector unit 10 is not greater than 1 μm.

[0122] Specifically, the size of a single detector unit in the photosensitive area of ​​the image acquisition chip is preferably 0.9 μm, and the number of detector units is preferably 14 million, in order to ensure full field of view and high resolution when imaging liquid-based cell samples.

[0123] As a preferred embodiment, the detector unit 10 may be a semi-floating gate transistor, a composite dielectric gate photodetector, a dual-device photodetector based on a composite dielectric gate, or a split-gate MOSFET imaging detector.

[0124] Specifically, when the detector unit uses a semi-floating gate transistor, the semi-floating gate transistor structure disclosed in the literature (Wang P, Lin X, Liu L, et al. A semi-floating gate transistor for low-voltage ultrafast memory and sensing operation.[J]. Science (New York, NY), 2013, 341(6146):640-643.) can be used, or the semi-floating gate transistor structure of Chinese patent CN201410201614.6 can be used, which includes a P-type semiconductor substrate, an N+ type source formed in the semiconductor substrate by ion implantation, and a large N+ type drain formed by two-step ion implantation. For example... Figure 7 As shown, a bottom dielectric layer, a semi-floating gate, a top dielectric layer, and a control gate are sequentially arranged on the semiconductor substrate. The bottom dielectric layer covers the upper surface of the P-type substrate and is positioned between the source and drain. The semi-floating gate layer covers the upper surface of the bottom dielectric layer, and a groove is etched in the middle of the bottom dielectric layer to allow direct contact between the semi-floating gate layer and the drain. Traditional floating gate transistors use an applied high voltage to control electron tunneling through the insulating dielectric layer for erasure and write operations. In contrast, the semi-floating gate transistor utilizes the quantum tunneling effect of a silicon-based TFET and employs a PN junction diode to replace the traditional silicon oxide data erasure window.

[0125] Specifically, when the detector unit 10 uses a composite dielectric gate photodetector, the photosensitive composite dielectric gate MOSFET detector disclosed in Chinese Patent CN200910024504.6 can be used. For example... Figure 8 As shown, the photodetector includes a P-type semiconductor substrate. A bottom dielectric layer, a charge storage layer, a top dielectric layer, and a control gate layer are sequentially disposed on top of the semiconductor substrate. N-type source and drain electrodes are formed in the semiconductor substrate through ion implantation doping, and the source and drain electrodes are respectively disposed on opposite sides of the bottom dielectric layer. By applying a gate voltage greater than the threshold voltage to the control gate, the voltage difference between the source and drain is zero. A large voltage difference between the P-type substrate and the source terminal creates a relatively wide depletion region on the substrate. Cold electrons generated in the substrate are accelerated towards the gate under the influence of the electric field in the depletion region, gaining energy. When this energy exceeds the potential barrier between the substrate and the bottom dielectric layer, the electrons directly cross this barrier and enter the bottom dielectric layer. Under the influence of the electric field of the bottom dielectric layer, they move towards the gate at a high speed, generating an injection current at the gate.

[0126] Specifically, when the detector unit adopts a dual-device photosensitive detection unit based on a composite dielectric grating, it can specifically adopt the dual-device photosensitive detection unit based on a composite dielectric grating disclosed in Chinese Patent CN201610592997.3, such as... Figure 11 and Figure 12 As shown, the dual-device photosensitive detection unit includes a photosensitive control gate and a readout control gate, which respectively have photosensitive and information readout functions. This dual-device photosensitive detection unit comprises two parts: a composite dielectric gate MOS-C and a composite dielectric gate MOSFET, both formed on the same P-type semiconductor substrate. The composite dielectric gate MOS-C part is used for photosensitive sensing, and the composite dielectric gate MOSFET part is used for information readout. Figure 9 As shown, the composite dielectric gate MOS-C portion includes a charge-coupled layer, a top dielectric layer, and a control gate sequentially stacked on a P-type semiconductor substrate, wherein an N-type injection layer is provided in the P-type semiconductor substrate; as shown Figure 10 As shown, the N-type injection layer is disposed below and in contact with the charge-coupled layer. The composite dielectric gate MOSFET includes a bottom dielectric layer, a charge-coupled layer, a top dielectric layer, and a control gate sequentially stacked on a P-type semiconductor substrate. A threshold adjustment injection region is provided in the substrate below the bottom dielectric layer. In the P-type semiconductor substrate, the N-type injection layer is separated from the N-type source region and the N-type drain region by a shallow trench isolation region and a P+ type injection region. When the dual-device photosensitive detection unit is working, a zero bias voltage is applied to the control gate, and a negative bias voltage pulse is applied to the substrate to form a depletion layer in the substrate. When light is incident on the depletion layer, photons are absorbed by the semiconductor, generating photoelectrons. Driven by the gate voltage, the photoelectrons move to the interface between the substrate and the gate oxide and accumulate at the interface, causing a change in the threshold voltage of the readout transistor, thereby characterizing the number of photoelectrons and converting the optical signal into a quantifiable electrical signal.

[0127] Specifically, the detector unit can also employ a split-gate MOSFET imaging detector, such as the split-gate MOSFET imaging detector disclosed in Chinese Patent CN201210349285.0. Figure 13As shown, the imaging detector structure includes a P-type semiconductor substrate, with two layers of insulating dielectric material and a control gate disposed on top of the substrate. A charge storage layer is disposed between the bottom and top dielectric layers. At least one location on the control gate or substrate has a transparent or semi-transparent window to allow the detector to detect light within a specific wavelength range. A selection gate is disposed on each side of the control gate, with a bottom dielectric layer between the selection gate and the substrate. The insulating dielectric layer at the bottom of the selection gate has the same material and thickness as the bottom dielectric layer at the bottom of the control gate. An N-type semiconductor region is disposed on the P-type substrate surrounding the substrate controlled by the two selection gates, forming the source and drain of a split-gate MOSFET. The two selection gates are located on both sides of the control gate, and the selection gate, control gate, and charge storage layer are separated by an insulating dielectric material, which also isolates the substrate controlled by the control gate from the source and drain of the imaging detector. The top dielectric layer in contact with the control gate is a material that prevents the charge stored in the charge storage layer from leaking into the control gate. The bottom dielectric layer in contact with the substrate P-type semiconductor material effectively isolates the substrate channel under the control gate from the charge storage layer. When the gate voltage is high enough or the incident photon energy is high, electrons in the channel are swept into the charge storage layer. A positive bias pulse is applied to the control gate, a negative bias pulse is applied to the substrate, and a negative bias pulse is applied to both select gates. This forms a depletion layer in the substrate under the control gate. When light is incident on the depletion layer and photons are absorbed by the semiconductor, photoelectrons are generated. Driven by the gate voltage, the photoelectrons move to the interface between the channel and the bottom dielectric layer. Because a negative bias is applied to both selection gates, a high electron barrier is formed in the substrate controlled by the selection gates. This high electron barrier effectively isolates the substrate controlled by the control gates from the N-type source and drain, ensuring that photoelectrons collected in the substrate depletion layer do not leak towards the source and drain. Simultaneously, electrons in the source and drain are also prevented from entering the substrate depletion layer by this high barrier. When the positive bias applied to the control gates is sufficiently large, photoelectrons collected in the substrate depletion layer will enter the charge storage layer via FN tunneling. If the incident photon energy is high enough, exceeding the bandgap between the semiconductor and the underlying dielectric layer, photoelectrons can enter the charge storage layer via direct tunneling. During the photoelectron collection stage, the source and drain can be appropriately positively biased, or they can be left floating.

[0128] In a preferred embodiment, the microscopic imaging device further includes:

[0129] An LED light source is mounted on the microscope imaging device;

[0130] An image processor is used to process the acquired image information. The photosensitive area of ​​the image acquisition chip is connected to the input terminal of the image processor through multiple pins.

[0131] A monitor, whose input is connected to the output of an image processor, is used to display processed image information.

[0132] Specifically, in this embodiment, when performing bright-field photography, an LED light source is needed to increase the brightness of the environment in which the liquid-based cell sample is located. After the light source illuminates the liquid-based cell sample, it generates a projection and is recorded by the photosensitive area, so that the microscopic image acquired by the photosensitive area of ​​the image acquisition chip is clearer.

[0133] It should be noted that when only the lower surface of the sample loading container is transparent, the LED light source is placed inside the sample loading container so that the light generated by the LED light source can illuminate the liquid-based cell sample; when the entire sample loading container is completely transparent, the LED light source is placed above the sample loading container so that the light generated by the LED light source passes through the sample loading container and illuminates the liquid-based cell sample.

[0134] In a preferred embodiment, the microscopic imaging device further includes:

[0135] An LED light source is mounted on the microscope imaging device;

[0136] A light shield is designed to fit the size of the LED light source, and the size of the light shield is also compatible with the size of the sample loading container, so that the light shield can cover the sample loading container.

[0137] An image processor is used to process the acquired image information. The photosensitive area of ​​the image acquisition chip is connected to the input terminal of the image processor through multiple pins.

[0138] A monitor, whose input is connected to the output of an image processor, is used to display processed image information.

[0139] Specifically, LED light sources are not required for dark-field imaging. Before injecting the liquid-based cell sample into the sample loading container, it can be labeled with a luciferase gene. The labeled liquid-based cell sample is then mixed with a luciferin substrate and injected into the sample loading container. The entire sample loading container is then encased in a light shield to isolate it from ambient light, creating a completely dark environment. The purpose of this is to image the spontaneous fluorescence of the liquid-based cell sample. The light shield material can preferably be opaque plastic, opaque metal, or other opaque polymer materials.

[0140] Specifically, after the image processor receives the microscopic image formed by the autofluorescence of the liquid-based cell sample, it removes or suppresses noise in the microscopic image through an internally preset image processing algorithm, and optimizes image data with poor imaging effect. For example, it performs image enhancement processing on image data with weak fluorescence signal, pseudo-color coloring processing on image data with blurred edges, and image segmentation processing on image data with low signal-to-noise ratio.

[0141] Specifically, the image processor can also be connected to a display, which receives the image data processed by the image processor and displays the final microscopic imaging results. Based on the imaging results displayed on the display, the microscopic image can be deleted, reduced, enlarged, rotated, selected for specific areas, and labeled with targets, facilitating further observation and analysis of liquid-based cell samples by the testing personnel.

[0142] Specifically, the image processor or display can also be equipped with an image storage device to establish an image database, storing image data and analysis results processed by the image processor. The image storage device contains a database, and image data can be stored locally or uploaded to a cloud server via a network. It also allows for searching, adding, deleting, modifying, and backing up image data within the database, facilitating subsequent data management by staff.

[0143] Example 2

[0144] The second embodiment of the present invention provides a microscopic imaging device for liquid-based cell samples, such as... Figure 1 As shown, it includes:

[0145] A photosensitive area 1 of an image acquisition chip is used to acquire image information of a liquid-based cell sample;

[0146] At least one sample loading container 2 is disposed on the photosensitive area 1 of the image acquisition chip, and the lower surface of the sample loading container 2 is attached to the upper surface of the photosensitive area 1 of the image acquisition chip. The lower surface of the sample loading container 2 is transparent. The sample loading container 2 is used to load liquid-based cell samples.

[0147] Each sample loading container 2 includes:

[0148] A hollow cavity 20;

[0149] Two through holes 21 are symmetrically arranged on both sides of the sample loading container 2 and are respectively connected to both ends of the hollow cavity 20. The two through holes 21 are located above the lower surface of the sample loading container 2 and are used for inputting and outputting liquid-based cell samples, respectively.

[0150] A platform 3 has a recess 30 on its upper surface, and the photosensitive area 1 of the image acquisition chip is disposed in the recess 30.

[0151] The sample loading container 2 has a protrusion 22 on the side facing the photosensitive area 1 of the image acquisition chip. The bottom of the protrusion 22 is adapted to the size of the recess 30 so that the protrusion 22 is fitted into the recess 30, and the lower surface of the protrusion 22 is in contact with the photosensitive area 1 of the image acquisition chip.

[0152] like Figure 17 As shown, the photosensitive area 1 of the image acquisition chip includes a plurality of pins 11, which are led out from both sides of the recess 30.

[0153] Specifically, the photosensitive area 1' of the image acquisition chip includes a plurality of pins 11' for connecting to external devices such as an image processor, etc. Figure 16 As shown, in the prior art, the pins 11' of the photosensitive area 1' of the image acquisition chip are led out from both sides of the photosensitive area 1' of the image acquisition chip. After the pins are packaged, the upper surface of the packaged pins is flush with the surface of the photosensitive area 1'. Figure 16 The lower surface of the sample loading container 2' shown is flush with the bottom surface, resulting in a certain gap between the sample loading container 2' and the photosensitive area 1' of the image acquisition chip, which leads to poor quality of the acquired microscopic images.

[0154] Furthermore, to address the aforementioned technical problems, this embodiment proposes a novel pin packaging structure, such as... Figure 4 As shown, since the sample loading container 2 in this embodiment has a protrusion 22 and the protrusion 22 is in the shape of an inverted trapezoid, a certain space is reserved on both sides of the protrusion 22, so that the pin 11 can be led out from the space on both sides. After the pin 11 is encapsulated, even if the encapsulated pin is flush with the lower surface of the sample loading container 2', it will not affect the image acquisition.

[0155] Example 3

[0156] The third embodiment of the present invention provides a microscopic imaging device for liquid-based cell samples, such as... Figure 14 As shown, it includes:

[0157] A photosensitive area 1 of an image acquisition chip is used to acquire image information of a liquid-based cell sample;

[0158] Two sample loading containers 2 are set on the photosensitive area 1 of the image acquisition chip, and the lower surface of each sample loading container 2 is attached to the upper surface of the photosensitive area 1 of the image acquisition chip. The lower surface of the sample loading container 2 is transparent. The sample loading container 2 is used to load liquid-based cell samples.

[0159] Each sample loading container 2 includes:

[0160] A hollow cavity 20;

[0161] Two through holes 21 are symmetrically arranged on both sides of the sample loading container 2 and are respectively connected to both ends of the hollow cavity 20. The two through holes 21 are located above the lower surface of the sample loading container 2 and are used for inputting and outputting liquid-based cell samples, respectively.

[0162] A platform 3 has a recess 30 on its upper surface, and the photosensitive area 1 of the image acquisition chip is disposed in the recess 30.

[0163] The sample loading container 2 has a protrusion 22 on the side facing the photosensitive area 1 of the image acquisition chip. The bottom of the protrusion 22 is adapted to the size of the recess 30 so that the protrusion 22 is fitted into the recess 30, and the lower surface of the protrusion 22 is in contact with the photosensitive area 1 of the image acquisition chip.

[0164] Two sample loading containers 2 are arranged side by side in the recess 30 of the loading platform 3.

[0165] Specifically, such as Figure 14 As shown, the two sample loading containers 2 are connected in parallel, and the photosensitive area 1 of one image acquisition chip can be used to detect the liquid-based cell samples in the two sample loading containers 2 at the same time; or the photosensitive areas 1 of two image acquisition chips can be set at the bottom of the recess 30 to detect the liquid-based cell samples in the two sample loading containers 2 respectively.

[0166] It should be noted that the arrangement of two parallel sample loading containers 2 in this embodiment is only a preferred embodiment. More sample loading containers and / or more photosensitive areas 1 of the image acquisition chip can be set according to actual needs. The number of sample loading containers 2 and photosensitive areas 1 of the image acquisition chip should not be regarded as a limitation of the present invention.

[0167] Example 4

[0168] The fourth embodiment of the present invention provides a microscopic imaging method for liquid-based cell samples, applicable to the microscopic imaging apparatus in any of the above embodiments, such as... Figure 18 As shown, the specific steps include:

[0169] Step S1: Dilute the liquid-based cell sample to form a diluent and inject it into the hollow cavity through one of the through holes of the sample loading container;

[0170] Step S2: Use the photosensitive area of ​​the image acquisition chip to acquire images of the diluent inside the hollow cavity to form image information;

[0171] Step S3: The image processor optimizes the image information.

[0172] Step S4: Display the optimized image information on the monitor.

[0173] Specifically, in this embodiment, such as Figure 1 As shown, the microscopic imaging device includes a photosensitive area 1 of an image acquisition chip and a sample loading container 2. The lower surface of the sample loading container 2 is completely attached to the upper surface of the photosensitive area 1 of the image acquisition chip, and the shape and size of the lower surface of the sample loading container 2 are adapted to the photosensitive area 1 of the image acquisition chip. The bottom of the sample loading container 2 is completely transparent, so that the liquid-based cell sample at the bottom of the sample loading container 2 can be completely exposed within the acquisition range of the photosensitive area 1 of the image acquisition chip, thereby realizing full-field microscopic observation and analysis. At the same time, since the lower surface of the sample loading container 2 is completely attached to the upper surface of the photosensitive area 1 of the image acquisition chip, the distance between the two is shortened, so as to reduce the influence of light refraction on the microscopic imaging results during subsequent image acquisition, effectively improving the quality of the acquired images.

[0174] Specifically, the liquid-based cell samples in this embodiment are exfoliated cells from mammalian urine or fecal dilution, pleural or peritoneal fluid, cerebrospinal fluid, sputum, or tracheal mucus, or exfoliated cells from mammalian oral or gastric mucosa or cervical scrapings, or blood cells or circulating tumor cells from mammalian blood.

[0175] Furthermore, in this embodiment, the liquid-based cell sample is preferably human blood. First, an appropriate amount of human blood sample is taken and diluted. Then, the diluent is injected into the hollow cavity 20 through the through-hole 21 on one side. After the diluent fills the bottom of the sample loading container, the photosensitive area 1 of the image acquisition chip is used to start acquiring a microscopic image of the liquid-based cell sample.

[0176] In a preferred embodiment, an LED light source can be pre-positioned directly above the photosensitive area 1 of the image acquisition chip. During bright-field imaging, after the diluent fills the bottom of the sample loading container, the LED light source is turned on, and the photosensitive area 1 of the image acquisition chip is used to begin acquiring microscopic images of the liquid-based cell sample, such as... Figure 15 The image shown is a microscopic imaging result of a diluted human blood sample. The microscopic imaging method described above can obtain a full-field microscopic image, in which a large number of red blood cells are clearly visible.

[0177] In a preferred embodiment, the liquid-based cell sample can be stained before being injected into the sample loading container 2. Because cells are small and colorless / transparent, the contrast between the cells and the background is very low, making it difficult to clearly see the cell morphology and identify certain cell structures. Therefore, in this embodiment, the cells are stained first. The contrast effect of the color enhances the observation of different parts of the cells, allowing for clearer observation and research. Furthermore, some staining methods can also be used to identify different cell groups.

[0178] In a preferred embodiment, step S1 further includes:

[0179] Step S11: Label the dilution solution using a luciferase gene;

[0180] Step S12: The labeled diluent is mixed with a fluorescein substrate.

[0181] Specifically, during dark-field imaging, there is no need to turn on the LED light source. Before injecting the liquid-based cell sample into the sample loading container, it can be labeled with a luciferase gene. The labeled liquid-based cell sample is then mixed with a luciferin substrate and injected into the sample loading container. The entire sample loading container is then covered with a light shield to isolate it from ambient light, creating a completely dark environment. The purpose is to use the spontaneous fluorescence of the liquid-based cell sample for imaging. The light shield material can preferably be opaque plastic, opaque metal, or other opaque polymer materials.

[0182] As a preferred embodiment, in step S2, before image acquisition, a light shield is used to seal the sample loading container so that image acquisition can be performed in a dark environment.

[0183] Specifically, after the image processor receives the microscopic image formed by the autofluorescence of the liquid-based cell sample, it removes or suppresses noise in the microscopic image through an internally preset image processing algorithm, and optimizes image data with poor imaging effect. For example, it performs image enhancement processing on image data with weak fluorescence signal, pseudo-color coloring processing on image data with blurred edges, and image segmentation processing on image data with low signal-to-noise ratio.

[0184] Example 5

[0185] This invention also provides a microfluidic chip for liquid-based cell sample microscopic imaging, such as... Figure 19-23As shown, the device includes a sample chamber 100 for loading liquid-based cell samples and an imaging chip 200 for image acquisition. The upper surface of the imaging chip 200 is attached to the lower surface of one or more of the sample chambers 100. The sample chamber 100 includes a liquid inlet 300, an intermediate cavity 600, and a liquid outlet 400. The liquid inlet 300 and the liquid outlet 400 are respectively connected to the intermediate cavity 600. The sample chamber 100 has a trapezoidal boss 500 structure at its bottom, and the intermediate cavity 600 is located on the trapezoidal boss 500.

[0186] The structure of the detector array of the imaging chip 200 is as follows: Figure 6 As shown, the detector array consists of a large number of identical individual detector units 10, and the structure of the individual detector unit 10 can be as follows: Figure 7 The semi-floating gate transistor shown can also be as follows: Figure 8 The composite dielectric grating photodetector shown can also be as follows: Figure 9 , Figure 10 , Figure 11 , Figure 12 The dual-device photosensitive detection unit based on a composite dielectric grating shown can also be as follows: Figure 13 The split-gate MOSFET imaging detector shown is illustrated. The detector array used in the imaging chip 2 of this embodiment is as follows... Figure 9 The dual-device photosensitive detector array based on a composite dielectric grating shown has an imaging chip 200 with a single detector unit size of 0.9 μm and a detector array size of 14 million, which is used to ensure a large field of view and high resolution when imaging liquid-based cell samples.

[0187] The thickness of the intermediate cavity 600 is between 120 μm.

[0188] The thickness from the upper surface to the lower surface of the sample cavity 100 is 5 mm.

[0189] The trapezoidal boss 500 has a apex angle of 120° and a thickness of 350μm.

[0190] The intermediate cavity 600 is rectangular.

[0191] The sample chamber 100 is completely transparent and is made of K9 optical glass.

[0192] The following is a brief description of the microscopic imaging process of liquid-based cell samples obtained using the microscopic imaging apparatus of this embodiment:

[0193] First, take an appropriate amount of human blood sample diluent and inject it into the intermediate cavity 600 through the inlet 300;

[0194] Then, turn on the LED light source directly above the microfluidic chip, and the imaging chip 200 begins to record the projected microscopic image of the cell sample, thus obtaining the microscopic imaging result of the diluted human blood sample. A full-field microscopic image can be obtained, and a large number of red blood cells are clearly visible.

[0195] Example 6

[0196] A microfluidic chip for microscopic imaging of liquid-based cell samples, such as Figure 20-24 As shown, the device includes a sample chamber 100 for loading liquid-based cell samples and an imaging chip 200 for image acquisition. The upper surface of the imaging chip 200 is attached to the lower surface of one or more of the sample chambers 100. The sample chamber 100 includes a liquid inlet 300, an intermediate cavity 600, and a liquid outlet 400. The liquid inlet 300 and the liquid outlet 400 are respectively connected to the intermediate cavity 600. The sample chamber 100 has a trapezoidal boss 500 structure at its bottom, and the intermediate cavity 600 is located on the trapezoidal boss 500.

[0197] The structure of the detector array of the imaging chip 200 is as follows: Figure 6 As shown, the detector array consists of a large number of identical individual detector units 10, and the structure of the individual detector unit 10 can be as follows: Figure 7 The semi-floating gate transistor shown can also be as follows: Figure 8 The composite dielectric grating photodetector shown can also be as follows: Figure 9 , Figure 10 , Figure 11 , Figure 12 The dual-device photosensitive detection unit based on a composite dielectric grating shown can also be as follows: Figure 13 The split-gate MOSFET imaging detector shown is an example. The detector array used in the imaging chip 200 of this embodiment is as follows. Figure 9 The dual-device photosensitive detector array based on a composite dielectric grating shown has an imaging chip 200 with a single detector unit size of 1 μm and a detector array size of 18 million, which is used to ensure a large field of view and high resolution when imaging liquid-based cell samples.

[0198] The thickness of the intermediate cavity 600 is 170 μm.

[0199] The thickness from the upper surface to the lower surface of the sample cavity 100 is 10 mm.

[0200] The trapezoidal boss 500 has a apex angle of 150° and a thickness of 500μm.

[0201] The intermediate cavity 600 is a rounded parallelogram.

[0202] The sample chamber 100 is completely transparent and is made of K9 optical glass.

[0203] The following is a brief description of the microscopic imaging process of liquid-based cell samples obtained using the microscopic imaging apparatus of this embodiment:

[0204] First, human circulating tumor cells labeled with the luciferase gene were mixed with a luciferin substrate to obtain an autofluorescent sample solution.

[0205] Next, take an appropriate amount of diluent for the autofluorescent human circulating tumor cell sample solution and inject it into the intermediate cavity 600 through the inlet 300.

[0206] Then, a light shield is placed over the device to isolate it from the surrounding light, providing a dark environment for the imaging process. The imaging chip then begins to record the projected microscopic image of the autofluorescent cell sample, resulting in a full-field microscopic image.

[0207] The above embodiments are merely preferred embodiments of the present invention. Any simple modifications, alterations, and substitutions made to the above embodiments based on the technical essence of the present invention shall fall within the scope of the technical solution of the present invention.

[0208] The beneficial effects of the technical solution of this invention are as follows:

[0209] (1) Compared with traditional optical microscopes, the microscopic imaging device in this invention can replace the traditional method of manually making glass slides, and directly use the photosensitive area of ​​the image acquisition chip to record the microscopic imaging results of liquid-based cell samples, which simplifies the operation steps of detection and analysis, and can avoid inaccurate detection results due to insufficient experience of the detection personnel in making glass slides.

[0210] (2) Through the photosensitive area of ​​the image acquisition chip, high-resolution microscopic images of the entire field of view can be directly acquired without the need for magnification by an optical lens. Compared with the prior art of sampling microscopic observation by moving the microscope objective lens to extract several fields of view, the microscopic imaging in this invention has higher accuracy and stability.

[0211] (3) The photosensitive area of ​​the image acquisition chip is set in the recess of the carrier platform, and a sample loading container with a protrusion is designed. The protrusion is embedded in the recess of the carrier platform, so that the lower surface of the sample loading container is in contact with the photosensitive area of ​​the image acquisition chip, which shortens the distance between the photosensitive area of ​​the image acquisition chip and the liquid-based cell sample, making the photosensitive area of ​​the image acquisition chip closer to the liquid-based cell sample, thereby improving the quality of the microscopic image.

[0212] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A microscopic imaging apparatus for a liquid-based cell sample, characterized by, The application relates to a microscopic imaging device. The device comprises: a photosensitive area of an image acquisition chip, which is used for acquiring image information of a liquid-based cell sample; at least one sample loading container, which is arranged on the photosensitive area of the image acquisition chip, and the lower surface of the sample loading container is attached to the upper surface of the photosensitive area of the image acquisition chip, the lower surface of the sample loading container is transparent, and the sample loading container is used for loading the liquid-based cell sample; each sample loading container comprises: a hollow cavity; two through holes, which are symmetrically arranged on the two sides of the sample loading container and respectively communicate with the two ends of the hollow cavity, the positions of the two through holes are higher than the lower surface of the sample loading container, and the two through holes are respectively used for inputting and outputting the liquid-based cell sample; the shape and size of the lower surface of the sample loading container are matched with the shape and size of the photosensitive area of the image acquisition chip; the device comprises a loading platform, the upper surface of the loading platform has a recess, and the photosensitive area of the image acquisition chip is arranged in the recess; 2. The microscopic imaging device of claim 1, wherein, one side of the sample loading container, which faces the photosensitive area of the image acquisition chip, has a protruding part, the bottom of the protruding part is matched with the size of the recess, so that the protruding part is clamped in the recess, and the lower surface of the protruding part is attached to the photosensitive area of the image acquisition chip.

3. The microscopic imaging device of claim 1, wherein, The protruding part is an inverted trapezoid.

4. The microscopic imaging device of claim 1, wherein, The two through holes are respectively higher than the upper surface of the protruding part.

5. The microscopic imaging device of claim 1, wherein, The thickness of the hollow cavity is 50-200 mu m.

6. The microscopic imaging device of claim 1, wherein, The whole sample loading container is transparent.

7. The microscopic imaging device of claim 1, wherein, The angle of the top corner of the protruding part is 90-180 degrees.

8. The microscopic imaging device of claim 1, wherein, The thickness of the protruding part is not less than 200 mu m.

9. The microscopic imaging device of claim 1, wherein, The shape of the hollow cavity is oval, circular, rectangular or round-cornered parallelogram.

10. The microscopic imaging device of claim 1, wherein, The sample loading container is made of transparent glass or transparent organic polymer.

11. The microscopic imaging device of claim 1, wherein, The liquid-based cell sample is first labeled by a luciferase gene, then mixed with a luciferin substrate and then enters the sample loading container.

12. The microscopic imaging device of claim 11, wherein, An LED light source is arranged above the microscopic imaging device.

13. The microscopic imaging device of claim 1, wherein, The microscopic imaging device further comprises a light shield cover which is matched with the LED light source, and the size of the light shield cover is matched with the size of the sample loading container, so that the light shield cover can wrap the sample loading container.

14. The microscopic imaging apparatus according to claim 13, characterized in that, The detector array of the image acquisition chip comprises a plurality of detector units, and the number of the detector units is not less than 10 million.

15. The microscopic imaging device of claim 1, wherein, The size of each detector unit is not greater than 1 mu m.

16. The microscopic imaging device of claim 15, wherein, The photosensitive area of the image acquisition chip comprises a plurality of pins which are respectively led out from the two sides of the recess.

17. The microscopic imaging device of claim 16, wherein, The microscopic imaging device further comprises an image processor, the photosensitive area of the image acquisition chip is connected to the input end of the image processor through a plurality of pins, and the image processor is used for processing the acquired image information.

18. The microscopic imaging device of claim 1, wherein, The microscopic imaging device further comprises a display, the input end of the display is connected to the output end of the image processor, and the display is used for displaying the processed image information. The microscopic imaging device comprises two sample loading containers, and the two sample loading containers are arranged side by side in the recess of the loading platform.

19. A microscopic imaging method for liquid-based cell samples, characterized in that... The application is applied to the microscopic imaging device as claimed in any one of claims 1-18, and specifically comprises the following steps: Step S1: diluting the liquid-based cell sample to form a diluent, and injecting the diluent into the hollow cavity through one of the through holes of the sample loading container; Step S2: using the photosensitive area of the image acquisition chip to acquire the image of the diluent in the hollow cavity to form the image information; Step S3: the image processor in the microscopic imaging device optimizes the image information; Step S4: displaying the optimized image information through the display.

20. The method of microscopic imaging according to claim 19, wherein, The step S1 further comprises: Step S11: using a luciferase gene to label the diluent; Step S12: mixing the labeled diluent with a luciferin substrate.

21. The method of microscopic imaging according to claim 19, wherein, In the step S2, before the image acquisition, a light shield is used to seal the sample loading container to perform the image acquisition in a dark environment.

22. The method of microscopic imaging according to claim 19, wherein, The optimization process comprises image enhancement processing and / or pseudo-color coloring processing and / or image segmentation processing.

23. A microfluidic chip for microscopic imaging of a liquid-based cytological sample, comprising: The application relates to a microscopic imaging device for loading a liquid-based cell sample, and comprises a sample cavity (100) and an imaging chip (200) for image acquisition, the upper surface of the imaging chip (200) is attached to the lower surface of one or more sample cavities (100), the sample cavity (100) comprises an inlet (300), an intermediate cavity (600) and an outlet (400), the inlet (300) and the outlet (400) are respectively communicated with the intermediate cavity (600), the lower surface of the sample cavity (100) is in a trapezoidal boss (500) structure, and the intermediate cavity (600) is located on the trapezoidal boss (500); The upper surface of the loading platform has a recess, and the photosensitive area of the imaging chip is arranged in the recess; One side of the sample cavity towards the photosensitive area of the imaging chip has a protruding part, the bottom of the protruding part is matched with the size of the recess, so that the protruding part is clamped in the recess, and the lower surface of the protruding part is attached to the photosensitive area of the imaging chip; The process of using the microfluidic chip for microscopic imaging comprises the following steps: First, mixing the luciferase gene labeled human circulating tumor cells with the luciferin substrate to obtain a self-fluorescent sample solution; Second, taking a proper amount of diluent of the self-fluorescent human circulating tumor cell sample solution, and injecting the diluent into the intermediate cavity (600) through the inlet (300); Then, covering a light shield to isolate the whole microfluidic chip from the light of the surrounding environment, and providing a dark environment for the imaging process, and the imaging chip starts to record the projection microscopic image of the cell sample in the self-fluorescent sample solution to obtain a full-field microscopic image.

24. The microfluidic chip for microscopic imaging of liquid-based cytological samples of claim 23, wherein, The detector array of the imaging chip (200) adopts one of a semi-floating gate transistor array, a composite dielectric gate photosensitive detector array, a double-device photosensitive detector unit array based on a composite dielectric gate, or a split-gate MOSFET imaging detector array.

25. The microfluidic chip for microscopic imaging of liquid-based cytological samples of claim 24, wherein, The single detector unit (10) of the imaging chip (200) has a size of ≤1μm, and the detector array has a scale of ≥100 million.

26. The microfluidic chip for microscopic imaging of liquid-based cytological samples of claim 23, wherein, The intermediate cavity (600) has a thickness of between 50μm and 200μm.

27. The microfluidic chip of claim 23, wherein, The sample cavity (100) has a thickness of ≥1mm from its upper surface to its lower surface.

28. The microfluidic chip for microscopic imaging of liquid-based cytological samples of claim 23, wherein, The top angle of the trapezoidal boss (500) has an angle of between 90° and 180°, and the thickness of the trapezoidal boss (500) is ≥200μm.

29. The microfluidic chip for microscopic imaging of liquid-based cytological samples of claim 23, wherein, The intermediate cavity (600) has an elliptical, circular, rectangular or rounded rectangular parallelepiped shape.

30. The microfluidic chip for microscopic imaging of liquid-based cytological samples of claim 23, wherein, The sample cavity (100) is completely transparent, and is made of transparent glass or transparent organic polymer.

31. The microfluidic chip for microscopic imaging of liquid-based cytological samples of claim 23, wherein, The liquid-based cell sample is selected from one of the following: exfoliated cells in urine, fecal dilution, pleural effusion, cerebrospinal fluid, sputum or tracheal mucus of a mammal, or exfoliated cells of buccal, gastric mucosa or cervical scraping of a mammal, or blood cells, circulating tumor cells in blood of a mammal.

32. The microfluidic chip for microscopic imaging of liquid-based cytological samples of claim 31, wherein, The mammal refers to a human.

33. The microfluidic chip for microscopic imaging of liquid-based cytological samples of claim 31, wherein, The liquid-based cell sample is selected from one of the following: exfoliated cells in urine, fecal dilution, pleural effusion, cerebrospinal fluid, sputum or tracheal mucus of a mammal, or exfoliated cells of buccal, gastric mucosa or cervical scraping of a mammal, or blood cells, circulating tumor cells in blood of a mammal after being dyed.

34. The microfluidic chip for microscopic imaging of liquid-based cytological samples of claim 31, wherein, The liquid-based cell sample is selected from one of the following: exfoliated cells in urine, fecal dilution, pleural effusion, cerebrospinal fluid, sputum or tracheal mucus of a mammal, or exfoliated cells of buccal, gastric mucosa or cervical scraping of a mammal, or blood cells, circulating tumor cells in blood of a mammal after being dyed. The liquid-based cell sample is selected from one of the following: exfoliated cells in urine, fecal dilution, pleural effusion, cerebrospinal fluid, sputum or tracheal mucus of a mammal, or exfoliated cells of buccal, gastric mucosa or cervical scraping of a mammal, or blood cells, circulating tumor cells in blood of a mammal after being dyed.

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