CN112161962A - Microscopic imaging device and microscopic imaging method for accelerating cell sedimentation - Google Patents

Microscopic imaging device and microscopic imaging method for accelerating cell sedimentation Download PDF

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CN112161962A
CN112161962A CN202011070001.5A CN202011070001A CN112161962A CN 112161962 A CN112161962 A CN 112161962A CN 202011070001 A CN202011070001 A CN 202011070001A CN 112161962 A CN112161962 A CN 112161962A
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microscopic imaging
imaging apparatus
liquid
microfluidic
image
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杨程
卢大骅
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Nanjing Jiuchuan Science And Technology Co ltd
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Nanjing Weipaishi Semiconductor Technology Co ltd
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    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
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    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
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    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1404—Handling flow, e.g. hydrodynamic focusing
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
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    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1404—Handling flow, e.g. hydrodynamic focusing
    • G01N2015/1415—Control of particle position
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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Abstract

The invention discloses a microscopic imaging device and a microscopic imaging method for accelerating cell sedimentation, which comprise the following steps: the photosensitive area of the image acquisition chip is used for acquiring image information of the liquid-based cell sample; the microfluidic chip comprises at least one microfluidic cavity, a liquid-based cell sample and a liquid-based cell sample, wherein the microfluidic cavity is arranged on a photosensitive area of the image acquisition chip, the lower surface of the microfluidic cavity is attached to the upper surface of the photosensitive area of the image acquisition chip, the lower surface of the microfluidic cavity is transparent, and the microfluidic cavity is used for loading the liquid-based cell sample; each microfluidic chamber comprises: a hollow cavity; the two through holes are symmetrically arranged on two sides of the micro-flow chamber; the two conductive films cover the upper surface and the lower surface of the micro-flow chamber respectively, are transparent and have opposite polarities. The technical scheme of the invention has the beneficial effects that: the light-sensitive area of the image acquisition chip is closer to the liquid-based cell sample, so that the quality of the microscopic image is improved, and the electric field formed by the two conductive films can accelerate cell sedimentation.

Description

Microscopic imaging device and microscopic imaging method for accelerating cell sedimentation
Technical Field
The invention relates to the technical field of microscopic imaging, in particular to a microscopic imaging device and a microscopic imaging method for accelerating cell sedimentation.
Background
The microscopic imaging device is used for optically amplifying microbial tissues, and realizes the detection of liquid-based cell samples and morphological analysis through the observation and recording of a photosensitive area of an image acquisition chip. The conventional optical microscope suffers from the contradiction between visual field and resolution, that is, the higher the magnification of the microbial tissue is, the finer the image of the microbial tissue can be seen, but the visual field range can be observed to be reduced.
In the microscopic imaging device in the prior art, the structure of the counting cell is mostly adopted, for example, chinese patent CN201520807546.8 discloses a microscope, which comprises a microscope platform and a counting cell platform assembly, wherein the counting cell and an infusion tube communicated with the counting cell are arranged on the counting cell platform assembly, a frame is fixed on the microscope platform, and the counting cell platform assembly is located in the frame and is pressed on the microscope platform. The patent is based on a traditional optical microscope, and a counting cell is arranged below an objective lens, so that the traditional manual preparation of slides and smears is replaced. However, the method only solves the problems of complicated flaking and unstable quality, and still depends on the observation of the traditional optical microscope, the microscopic observation of the whole field of view cannot be realized, and only part of the field of view can be selected for sampling observation and analysis within the limited detection time range, so that the accuracy of detection and analysis is reduced; meanwhile, the conventional optical microscope has a large volume and is very expensive. In addition, chinese patent CN201811030915.1 discloses a bright field and fluorescence dual-mode microscopic imaging system, which adopts an image sensor chip to acquire an image of a sample to be detected. The microscopic imaging system comprises an image sensor chip and a micro-flow chamber, wherein the bottom of the micro-flow chamber is not directly contacted with the image sensor chip, but a light filter is arranged between the image sensor chip and the micro-flow chamber, and the image sensor chip can possibly generate light refraction in the process of collecting images, so that the quality of image collection is influenced.
Before the image sensor chip collects the cell image, the cells injected into the microfluidic chamber need to be stood, so that the cells naturally settle to a stable height, and focusing is facilitated. However, cells of different sizes end up stabilizing at slightly different heights in the liquid, and the time to wait for the cell suspension to settle naturally in the microfluidic chamber is long, and even after stabilization, there are still many cells at different heights. Therefore, a micro-imaging device with a function of accelerating cell sedimentation is needed to reduce the time of cell sedimentation so that the image sensor chip can rapidly acquire clear micro-images.
Disclosure of Invention
In view of the above problems in the prior art, a microscopic imaging apparatus and a microscopic imaging method for accelerating cell sedimentation are provided.
The specific technical scheme is as follows:
the invention includes a microscopic imaging device for accelerating cell sedimentation, comprising: the image acquisition chip comprises a photosensitive area and is used for acquiring image information of a liquid-based cell sample; the microfluidic chamber is arranged on the photosensitive area, the lower surface of the microfluidic chamber is attached to the upper surface of the photosensitive area, the lower surface of the microfluidic chamber is transparent, and the microfluidic chamber is used for loading the liquid-based cell sample;
each of the microfluidic chambers includes:
a hollow cavity;
the two through holes are symmetrically arranged on two sides of the micro-flow chamber and are respectively communicated with two ends of the hollow cavity, and the positions of the two through holes are higher than the lower surface of the micro-flow chamber and are respectively used for inputting and outputting the liquid-based cell sample;
the microscopic imaging apparatus further includes:
the two conductive films are respectively covered on the upper surface and the lower surface of the microfluidic chamber, the two conductive films are transparent and have opposite polarities, and the cells in the liquid-based cell sample are accelerated to settle to the bottom of the microfluidic chamber under the action of an electric field formed by the two conductive films.
Preferably, the micro-imaging device comprises a plurality of micro-flow chambers, and two pieces of the conductive thin films comprise:
the first conductive film comprises a plurality of first conductive sub-films, and each first conductive sub-film covers the upper surface of one corresponding micro-flow chamber;
and the second conductive film comprises a plurality of second conductive sub-films, and each second conductive sub-film covers the lower surface of one corresponding micro-flow chamber.
Preferably, when the liquid-based cell sample injected into the microfluidic chamber has negative charges, the polarity of the first conductive film is a negative electrode, and the polarity of the second conductive film is a positive electrode;
when the liquid-based cell sample injected into the micro-flow chamber is positively charged, the polarity of the first conductive film is positive, and the polarity of the second conductive film is negative.
Preferably, when the liquid-based cell sample in the microfluidic chamber is negatively charged, the polarity of the corresponding first conductive film is a negative electrode, and the polarity of the corresponding second conductive film is a positive electrode;
when the liquid-based cell sample in the microfluidic chamber is positively charged, the polarity of the corresponding first conductive film is a positive electrode, and the polarity of the corresponding second conductive film is a negative electrode.
Preferably, the material of the conductive film is tin-doped indium oxide or aluminum-doped zinc oxide.
Preferably, the device comprises a carrying platform, wherein the upper surface of the carrying platform is provided with a concave part, and the photosensitive area is arranged in the concave part.
Preferably, one surface of the micro-flow chamber facing the photosensitive area is provided with a protruding portion, and the bottom of the protruding portion is matched with the size of the recessed portion, so that the protruding portion is embedded in the recessed portion in a clamping manner, and the lower surface of the protruding portion is attached to the photosensitive area.
Preferably, the protrusion has an inverted trapezoidal shape.
Preferably, the two through holes are respectively higher than the upper surfaces of the protruding parts.
Preferably, the thickness of the hollow cavity is 50-200 μm.
Preferably, the microfluidic chamber is transparent as a whole.
Preferably, the angle of the apex angle of the protrusion is 90 to 180 °.
Preferably, the thickness of the protruding portion is not less than 200 μm.
Preferably, the hollow cavity is in the shape of an ellipse, a circle, a rectangle or a rounded parallelogram.
Preferably, the microfluidic chamber is made of transparent glass or transparent organic polymer.
Preferably, the liquid-based cell sample is labeled with a luciferase gene, and then mixed with a luciferin substrate to enter the microfluidic chamber.
Preferably, an LED light source is arranged above the microscopic imaging device.
Preferably, the micro-imaging device further comprises a light shield adapted to the LED light source, and the size of the light shield is adapted to the size of the micro-flow chamber, so that the light shield can wrap the micro-flow chamber.
Preferably, the photosensitive area of the image capturing chip includes a detector array, the detector array includes a plurality of detector units, and the number of the detector units is not less than 1 million.
Preferably, each of the detector cells has a size of no more than 1 μm.
Preferably, the photosensitive area of the image acquisition chip comprises a plurality of pins, and the pins are respectively led out from two sides of the concave part.
Preferably, the microscopic imaging device further comprises an image processor, the photosensitive region is connected with an input end of the image processor through a plurality of pins, and the image processor is used for processing the acquired image information.
Preferably, the microscopic imaging device further comprises a display, and an input end of the display is connected with an output end of the image processor and is used for displaying the processed image information.
Preferably, the microscopic imaging device comprises two microfluidic chambers, and the two microfluidic chambers are arranged in parallel in the concave portion of the stage.
The invention also comprises a microscopic imaging method, which is applied to the microscopic imaging device in the technical scheme 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 microfluidic chamber;
step S2, using the photosensitive region to perform image acquisition on the diluent in the hollow cavity to form the image information;
step S3, the image processor performs optimization processing on the image information;
and step S4, displaying the optimized image information through a display.
Preferably, the step S1 further includes:
step S11, labeling the diluted solution with a luciferase gene;
and step S12, mixing the marked diluent with a fluorescein substrate.
Preferably, in step S2, before the image acquisition, a light shield is used to seal the microfluidic chamber for the image acquisition in a dark environment.
Preferably, the optimization process includes an image enhancement process and/or a pseudo color shading process and/or an image segmentation process.
The technical scheme of the invention has the beneficial effects that: by adopting the microscopic imaging device in the embodiment of the invention, the lower surface of the microfluidic cavity is attached to the photosensitive area of the image acquisition chip, the distance between the photosensitive area and the liquid-based cell sample is shortened, and the photosensitive area is closer to the liquid-based cell sample, so that the quality of a microscopic image is improved; in addition, because the electrode films with opposite polarities are arranged on the upper surface and the lower surface of the microfluidic chamber, after the liquid-based cell sample is injected into the microfluidic chamber, the unidirectional electric field formed by the two electrode films accelerates the sedimentation speed of the cells so as to solve the technical problem of long sedimentation time caused by natural sedimentation, and the cells can be completely pushed to the bottom of the microfluidic chamber; in addition, the conductive film is made of transparent conductive materials, does not influence the transmission of light, and can be compatible with the existing microscopic imaging device.
Drawings
Embodiments of the present invention will be described more fully with reference to the accompanying drawings. The drawings are, however, to be regarded as illustrative and explanatory only and are not restrictive of the scope of the invention.
FIG. 1 is a schematic view of the overall configuration of a microscopic imaging apparatus in an embodiment of the present invention;
FIG. 2 is a diagram illustrating a structure of a pin package of a photosensitive region of an image capture chip according to the prior art;
FIG. 3 is a schematic diagram of a lead package of a photosensitive region of an image sensor chip according to an embodiment of the invention;
FIG. 4 is a schematic view of a microfluidic chamber at a first viewing angle in an embodiment of the present disclosure;
FIG. 5 is a schematic view of a microfluidic chamber at a second viewing angle in an embodiment of the present disclosure;
FIG. 6 is a side view of a microfluidic chamber in an embodiment of the present invention;
FIG. 7 is a top view of a microfluidic chamber according to an embodiment of the present invention;
FIG. 8 is a schematic diagram of a detector array of a photosensitive area of an image capture chip according to an embodiment of the present invention;
FIG. 9 is a schematic structural diagram of a semi-floating gate transistor in an embodiment of the present invention;
FIG. 10 is a schematic structural diagram of a composite dielectric grid photosensitive detector in an embodiment of the invention;
FIG. 11 is a first type of structure of a dual device photo-sensing detection unit based on a composite dielectric gate according to an embodiment of the present invention;
FIG. 12 is a second type of structure of a dual device photo-sensing detection unit based on a composite dielectric gate in an embodiment of the present invention;
FIG. 13 is a third type of structure of a dual device photo-sensing detection unit based on a composite dielectric gate in an embodiment of the present invention;
FIG. 14 shows a fourth type of structure of a dual device photo-sensing detection unit based on a composite dielectric gate according to an embodiment of the present invention;
FIG. 15 is a schematic structural diagram of a split-gate MOSFET imaging detector in an embodiment of the present invention;
FIG. 16 is a schematic structural diagram of a micro-imaging device having two micro-fluidic chambers in an embodiment of the present invention;
FIG. 17 shows the result of microscopic imaging of human blood sample diluent collected by the microscopic imaging apparatus according to the embodiment of the present invention;
FIG. 18 is a flow chart of steps of a method of microscopic imaging in an embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
The invention is further described with reference to the following drawings and specific examples, which are not intended to be limiting.
Example one
A first embodiment of the present invention provides a microscopic imaging apparatus for accelerating cell sedimentation, as shown in fig. 1, comprising:
the image acquisition chip comprises a photosensitive area 1 and is used for acquiring image information of a liquid-based cell sample;
the microfluidic cavity 2 is arranged on the photosensitive area 1, the lower surface of the microfluidic cavity 2 is attached to the upper surface of the photosensitive area 1, the lower surface of the microfluidic cavity 2 is transparent, and the microfluidic cavity 2 is used for loading liquid-based cell samples;
each microfluidic chamber 2 comprises:
a hollow cavity 20;
the two through holes 21 are symmetrically arranged on two sides of the microfluidic chamber 2 and are respectively communicated with two ends of the hollow cavity 20, and the positions of the two through holes 21 are higher than the lower surface of the microfluidic chamber 2 and are respectively used for inputting and outputting liquid-based cell samples.
The microscopic imaging apparatus further includes:
the two conductive films (201 and 202 shown in fig. 3) cover the upper surface and the lower surface of the microfluidic chamber 2, respectively, the two conductive films are transparent, and the polarities of the two conductive films are opposite, so that the cells in the liquid-based cell sample are accelerated to sink to the bottom of the microfluidic chamber 2 under the action of an electric field formed by the two conductive films.
Specifically, in the present embodiment, as shown in fig. 1, the micro-imaging device includes an image capturing chip and a micro-flow chamber 2, a lower surface of the micro-flow chamber 2 is completely attached to an upper surface of a photosensitive region 1 of the image capturing chip, the lower surface of the micro-flow chamber 2 is matched with the shape and the size of the photosensitive area 1 of the image acquisition chip, the bottom of the micro-flow chamber 2 is in a completely transparent state, so that the liquid-based cell sample at the bottom of the micro-flow chamber 2 can be completely exposed in the collection range of the photosensitive area 1 of the image collection chip, thereby realizing the microscopic observation and analysis of the full field of view and simultaneously, because the lower surface of the microflow chamber 2 is completely attached to the upper surface of the photosensitive area 1 of the image acquisition chip, the distance between the microflow chamber and the photosensitive area is shortened, the influence of light refraction on an imaging result is reduced, and the quality of an acquired image is effectively improved.
Specifically, the microfluidic chamber 2 includes two through holes 21, wherein a first through hole 21A is used for injecting the liquid-based cell sample, and after the image acquisition and analysis of the liquid-based cell sample are completed, the liquid-based cell sample is output from the microfluidic chamber 2 through a second through hole 21B on the other side. In addition, because the through holes 21 are arranged in parallel with the photosensitive area 1 of the image acquisition chip, when the micro-flow chamber 2 is cleaned, cleaning liquid can be input through the through hole on one side, and then the cleaning liquid is discharged through the through hole on the other side, so that the cleaning liquid is favorably discharged. The micro-flow chamber 2 can be repeatedly used by arranging the through holes on the two sides of the micro-flow chamber.
Specifically, in this embodiment, the microscopic imaging device includes at least one microfluidic chamber 2 for the flow of the liquid-based cell sample, and since the polarities of the two conductive films are opposite, a unidirectional electric field is formed in a region between the two conductive films, and the microfluidic chamber 2 is located in the unidirectional electric field, after the cell sample is injected into the microfluidic chamber 2, the cell is pushed to the bottom of the microfluidic chamber 2 under the action of the electric field, and compared with natural sedimentation, the sedimentation time can be effectively reduced.
The position of the conductive film is related to the polarity of the self-charge of the cell. The cells are generally negatively charged, so that the conductive film with the negative polarity is arranged on the upper surface of the microfluidic chamber 2, the conductive film with the positive polarity is arranged on the lower surface of the microfluidic chamber 2, and the liquid-based cell sample can accelerate towards the conductive film with the positive polarity after being injected into the microfluidic chamber 2, so that the sedimentation speed of the cells is increased. Similarly, if the cells have positive charges, the conductive film with the positive polarity can be arranged on the upper surface of the microfluidic chamber 2, and the conductive film with the negative polarity can be arranged on the lower surface of the microfluidic chamber 2, so that the cells are forced to be pushed to the bottom of the microfluidic chamber, and the problem that the cells do not settle or the settling speed is low is solved.
In a preferred embodiment, the two sheets of conductive film comprise:
the first conductive film comprises a plurality of first conductive sub-films, and each first conductive sub-film covers the upper surface of a corresponding micro-flow chamber;
and the second conductive film comprises a plurality of second conductive sub-films, and each second conductive sub-film covers the lower surface of one corresponding micro-flow chamber.
Specifically, a plurality of micro-flow chambers are usually arranged on the micro-imaging device, in this embodiment, it is preferable to arrange a conductive thin film for a region corresponding to each micro-flow chamber, and the upper surface of the micro-flow chamber can be provided with a plurality of conductive sub-thin films.
In a preferred embodiment, the conductive film is made of tin-doped indium Oxide (ITO). It should be noted that, the ITO material is only a preferred embodiment of the present invention, the present invention is not limited to the material of the conductive film, and other transparent conductive materials, such as Aluminum-doped Zinc oxide (AZO for short), may also be used, and the use of the transparent material does not affect the transmission of light, and avoids affecting the quality of the microscopic image.
As a preferred embodiment, as shown in fig. 1, the microscopic imaging device includes a stage 3, the stage 3 has a recess 30 on the upper surface, and the photosensitive region 1 is disposed in the recess 30;
one surface of the micro-flow chamber 2 facing the light-sensing region 1 of the image capturing chip is provided with a protruding portion 22, and the bottom of the protruding portion 22 is matched with the size of the recessed portion 30, so that the protruding portion 22 is embedded in the recessed portion 30, and the lower surface of the protruding portion 22 is attached to the light-sensing region 1 of the image capturing chip.
Specifically, in the present embodiment, as shown in fig. 1, the upper surface of the stage 3 has a recess 30, the light sensing region 1 of the image capturing chip is disposed in the recess 30, and the shape and size of the recess 30 match the light sensing region 1. As shown in fig. 6 and 7, the periphery of the groove portion 30 is used for encapsulating the leads of the photosensitive area 1 of the image capturing chip. It should be noted that the object stage 3 is a whole image capturing chip, and when the image capturing chip is packaged, the package glue is needed to fix the package of the tube shell to the peripheral area of the image capturing chip, and the peripheral area of the image capturing chip is protruded by such a packaging manner, so that the recess 30 is formed. The photosensitive area of the image capture chip is located in the recess 30, which causes the problem that the bottom of the microfluidic chamber in the prior art cannot directly contact the photosensitive area.
Specifically, as shown in fig. 4 and 5, the micro-flow chamber 2 in the present embodiment has a protruding portion 22, the protruding portion 22 is in an inverted trapezoid shape, and the shape and size of the protruding portion 22 are also adapted to the light-sensing region 1 of the image capturing chip, so that the protruding portion 22 is embedded in the recessed portion 30, and thus the lower surface of the protruding portion 22 is attached to the upper surface of the light-sensing region 1 of the image capturing chip, thereby shortening the distance therebetween and improving the imaging quality.
In this embodiment, the bottom of the protrusion 22 may be transparent, or the microfluidic chamber 2 may be completely transparent, and the transparent portion may be made of transparent glass or transparent organic polymer.
In a preferred embodiment, the thickness of the microfluidic chamber is not less than 1mm, preferably 5mm in this embodiment;
the thickness of the hollow cavity 20 is 50 to 200 μm, preferably 120 μm in this embodiment;
the thickness of the protruding portion 22 is not less than 200 μm, preferably 350 μm in the present embodiment;
the angle of the apex angle of the protrusion 22 is between 90 ° and 180 °, and preferably 120 ° in the present embodiment.
In a preferred embodiment, as shown in fig. 8, the photosensitive area 1 of the image capturing chip includes a detector array formed by a plurality of detector units 10, the number of the detector units 10 is not less than 1 million, and the size of each detector unit 10 is not greater than 1 μm.
Specifically, the size of the single detector unit of the photosensitive area of the image acquisition chip is preferably 0.9 μm, and the number of the detector units is preferably 1.4 million, so as to ensure the full field of view and high resolution in the microscopic imaging of the liquid-based cell sample.
As a preferred embodiment, the detector unit 10 can adopt a semi-floating gate transistor or a composite dielectric gate photosensitive detector or a composite dielectric gate-based dual-device photosensitive detection unit or a split-gate MOSFET imaging detector;
specifically, when the detector unit employs a semi-floating gate transistor, a 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 ultra-gate transistor and sensing operation [ J ]. Science (New York, n.y.),2013,341(6146): 640-643), or a semi-floating gate transistor structure of chinese patent CN201410201614.6, which includes a P-type semiconductor substrate in which an N + -type source is formed by ion implantation and a large N + -type drain is formed by two-step ion implantation, may be employed. As shown in fig. 9, a bottom dielectric layer, a semi-floating gate, a top dielectric layer and a control gate are sequentially disposed above the semiconductor substrate, the bottom dielectric layer covers the upper surface of the P-type substrate and is disposed between the source and the drain, the semi-floating gate layer covers the upper surface of the bottom dielectric layer, and a groove is formed in the middle of the bottom dielectric layer by etching, so that the semi-floating gate layer is in direct contact with the drain. The erase and write operation of the conventional floating gate transistor is controlled by applying a high voltage to control electron tunneling through the insulating dielectric layer, while the semi-floating gate transistor uses the quantum tunneling effect of TFET in the silicon body and uses a PN junction diode to replace the conventional silicon oxide data erase and write window.
In particular, when the detector unit 10 employs a composite dielectric gate photosensitive detector, a photosensitive composite dielectric gate MOSFET detector disclosed in chinese patent CN200910024504.6 may be employed. As shown in fig. 10, the photosensitive detector 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 over the semiconductor substrate, an N-type source and a drain are formed in the semiconductor substrate by ion implantation doping, and the source and the drain are respectively disposed on two sides of the bottom dielectric layer. The voltage difference between the source electrode and the drain electrode is 0 by applying a grid voltage which is larger than the threshold voltage on the control grid, and a large voltage difference is arranged between the P-type substrate and the source electrode to generate a relatively wide depletion region on the substrate, so that cold electrons generated in the substrate are accelerated towards the direction of the grid electrode under the action of an electric field in the depletion region to obtain energy, when the energy is larger than a potential barrier between the substrate and a bottom medium layer, the electrons directly cross the potential barrier to enter the bottom medium layer, and move towards the direction of the grid electrode at a high speed under the action of the electric field of the bottom medium layer to generate the injection current of the grid electrode.
Specifically, when the detector unit adopts a dual-device photosensitive detection unit based on a composite dielectric gate, the dual-device photosensitive detection unit based on a composite dielectric gate disclosed in chinese patent CN201610592997.3 may be specifically adopted, as shown in fig. 13 and 14, the dual-device photosensitive detection unit includes a photosensitive control gate and a read control gate, and has a photosensitive function and an information reading function respectively. The double-device photosensitive detection unit comprises a composite dielectric gate MOS-C part and a composite dielectric gate MOSFET part, wherein the composite dielectric gate MOS-C part and the composite dielectric gate MOSFET part are formed above the same P-type semiconductor substrate, the composite dielectric gate MOS-C part is used for sensitization, and the composite dielectric gate MOSFET part is used for reading information. As shown in fig. 11, the MOS-C portion of the composite dielectric gate includes a charge coupling layer, a top dielectric layer and a control gate sequentially stacked above a P-type semiconductor substrate, wherein an N-type injection layer is disposed in the P-type semiconductor substrate; as shown in fig. 12, the N-type injection layer is disposed below the charge coupling layer and contacts with the charge coupling layer, the composite dielectric gate MOSFET includes a bottom dielectric layer, a charge coupling layer, a top dielectric layer and a control gate sequentially stacked above the P-type semiconductor substrate, and a threshold adjustment injection region is disposed 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 electrode region and the N-type drain electrode region by arranging a shallow groove isolation region and a P + type injection region. When the dual-device photosensitive detection unit works, a control gate applies a bias voltage of 0, a substrate applies a negative bias voltage pulse, a depletion layer is formed in the substrate, when photons are absorbed by a semiconductor when light is incident into the depletion layer, photoelectrons are generated, the photoelectrons are driven by a gate voltage to move to an interface between the substrate and the gate oxide and gather at the interface, so that the threshold voltage of a reading transistor is changed, the number of the photoelectrons is represented, and an optical signal is converted into a quantifiable electric signal.
In particular, the detector unit may also employ a split-gate MOSFET imaging detector, such as the split-gate MOSFET imaging detector disclosed in chinese patent CN 201210349285.0. As shown in fig. 15, the structure of the imaging detector includes a P-type semiconductor substrate, two layers of insulating dielectric materials and a control gate are respectively disposed above the substrate, and a charge storage layer is disposed between a bottom dielectric layer and a top dielectric layer. At least one of the control gate or the substrate is provided with a transparent or translucent window to allow the detector to detect light in a wavelength range. And the two sides of the control grid are respectively provided with a selection grid, a bottom dielectric layer is arranged between the selection grid and the substrate, and the material and the thickness of the insulating dielectric layer at the bottom of the selection grid are the same as those of the bottom dielectric layer at the bottom of the control grid. N-type semiconductor regions are arranged on the peripheral P-type substrate of the substrate controlled by the two selection gates to form a source electrode and a drain electrode of the split-gate MOSFET. The two selection gates are arranged on two sides of the control gate, the selection gates, the control gate and the charge storage layer are separated by insulating medium materials, and a substrate controlled by the control gate is separated from a source electrode and a drain electrode of the imaging detector. The top dielectric layer in contact with the control gate is a material for preventing charges stored in the charge storage layer from being lost to the control gate, the bottom dielectric layer in contact with the substrate P-type semiconductor material can effectively isolate a substrate channel and the charge storage layer under the control of the control gate, and electrons in the channel can be swept into the charge storage layer when the gate voltage is high enough or the incident photon energy is high. And applying a positive bias pulse on the control gate, applying a negative bias pulse on the substrate, and simultaneously applying a negative bias pulse on the two selection gates, so that a depletion layer is formed in the substrate under the control of the control gate, and when photons are absorbed by the semiconductor when light is incident on the depletion layer, photoelectrons are generated and move to the interface of the channel and the bottom dielectric layer under the drive of the gate voltage. Because the two selection gates are applied with a negative bias voltage, a high electron barrier is formed in the substrate controlled by the selection gates, the substrate controlled by the control gates is effectively isolated from the N-type source electrode and the N-type drain electrode by the high electron barrier, the photoelectrons collected in the substrate depletion layer can not be lost towards the source electrode and the drain electrode, and meanwhile, the electrons in the source electrode and the drain electrode can not enter the substrate depletion layer due to the obstruction of the high electron barrier. When the positive bias applied by the control gate is large enough, the photoelectrons collected in the substrate depletion layer enter the charge storage layer by means of F-N tunneling; if the energy of the incident photons is high enough and is larger than the forbidden bandwidth of the semiconductor and the bottom dielectric layer, the photoelectrons can enter the charge storage layer by direct tunneling. During the photoelectron collection phase, the source and drain may be properly biased positively or floated directly.
As a preferred embodiment, the microscopic imaging apparatus further comprises:
the LED light source is arranged on the microscopic imaging device;
the photosensitive area of the image acquisition chip is connected with the input end of the image processor through a plurality of pins, and the image processor is used for processing acquired image information;
and the input end of the display is connected with the output end of the image processor and is used for displaying the processed image information.
Specifically, in this embodiment, when taking a bright field photograph, the LED light source is required to improve the brightness of the environment where the liquid-based cell sample is located, and the light source generates a projection after irradiating the liquid-based cell sample and is recorded by the photosensitive area, so that the microscopic image acquired by the photosensitive area of the image acquisition chip is clearer.
When only the lower surface of the microfluidic chamber is transparent, the LED light source is disposed inside the microfluidic chamber, so that light generated by the LED light source can irradiate on the liquid-based cell sample; when the microfluidic chamber is completely transparent, the LED light source is arranged above the microfluidic chamber, and light rays generated by the LED light source irradiate the liquid-based cell sample through the microfluidic chamber.
As a preferred embodiment, the microscopic imaging apparatus further comprises:
the LED light source is arranged on the microscopic imaging device;
the light shield is matched with the size of the LED light source, and the size of the light shield is matched with the size of the micro-flow chamber, so that the light shield can wrap the micro-flow chamber;
the image processor is used for processing the acquired microscopic image data;
and the input end of the display is connected with the output end of the image processor and is used for displaying the processed image data.
Specifically, the microscopic imaging device can support two acquisition modes of bright field shooting and dark field shooting. When dark field photographing is carried out, an LED light source is not needed. Before injecting the liquid-based cell sample into the microfluidic chamber, the luciferase gene can be labeled, and then the labeled liquid-based cell sample and the luciferin substrate are mixed and injected into the microfluidic chamber. The light shield encloses the entire microfluidic chamber so that the microfluidic chamber is in a completely dark environment aimed at imaging using the spontaneous fluorescence of the liquid-based cell sample. The light shield material may preferably be an opaque plastic or an opaque metal or other polymer material that is opaque. The image processor is used for carrying out optimization processing such as noise reduction on the microscopic image, so that the imaging effect is improved; the display is used for receiving the image data processed by the image processor and displaying the final microscopic imaging result, and the detecting personnel can adjust the parameters such as the size, the display mode and the like of the microscopic image according to the displayed imaging result of the display, so that the detecting personnel can further observe and analyze the liquid-based cell sample. The image processor or the display in this embodiment may further include an image memory for establishing an image database and storing the image data and the analysis result after passing through the image processor.
Example two
A second embodiment of the present invention provides a microscopic imaging apparatus for liquid-based cell samples, as shown in fig. 1, comprising:
the photosensitive area 1 of the image acquisition chip is used for acquiring image information of a liquid-based cell sample;
the microfluidic chip comprises at least one microfluidic chamber 2, a light sensing area 1 and a light-sensitive area, wherein the microfluidic chamber 2 is arranged on the light sensing area 1 of the image acquisition chip, the lower surface of the microfluidic chamber 2 is attached to the upper surface of the light sensing area 1 of the image acquisition chip, the lower surface of the microfluidic chamber 2 is transparent, and the microfluidic chamber 2 is used for loading a liquid-based cell sample;
each microfluidic chamber 2 comprises:
a hollow cavity 20;
the two through holes 21 are symmetrically arranged on two sides of the microfluidic chamber 2 and are respectively communicated with two ends of the hollow cavity 20, and the two through holes 21 are higher than the lower surface of the microfluidic chamber 2 and are respectively used for inputting and outputting liquid-based cell samples;
the upper surface of the object carrying platform 3 is provided with a concave part 30, and the photosensitive area 1 of the image acquisition chip is arranged in the concave part 30;
one surface of the micro-flow chamber 2 facing the light-sensing area 1 of the image acquisition chip is provided with a protruding part 22, the bottom of the protruding part 22 is matched with the size of the recessed part 30, so that the protruding part 22 is embedded in the recessed part 30, and the lower surface of the protruding part 22 is attached to the light-sensing area 1 of the image acquisition chip;
as shown in fig. 3, the photosensitive area 1 of the image capturing chip includes a plurality of leads 11, and the leads 11 are respectively led out from two sides of the recess 30.
Specifically, the light sensing region 1 'of the image capturing chip includes a plurality of pins 11' for connecting to external devices such as an image processor, as shown in fig. 2, in the prior art, the pins 11 'of the light sensing region 1' of the image capturing chip are led out from two sides of the light sensing region 1 'of the image capturing chip, and after the pins are packaged, the upper surfaces of the packaged pins are flush with the lower surface of the microfluidic chamber 2' shown in fig. 2, so that a certain gap is formed between the microfluidic chamber 2 'and the light sensing region 1' of the image capturing chip, and the quality of the captured microscopic image is poor.
Further, in view of the above technical problems, a novel pin packaging structure is provided in this embodiment, as shown in fig. 4, since the microfluidic cavity 2 in this embodiment has the protruding portion 22, and the protruding portion 22 is in an inverted trapezoid shape, so that a certain space is reserved on two sides of the protruding portion 22, the pin 11 can be led out from the spaces on two sides, and after the pin 11 is subsequently packaged, even if the packaged pin is flush with the lower surface of the microfluidic cavity 2', the image collection cannot be affected. In addition, the bottom of the protruding portion 22 is provided with a second conductive film 202, and a unidirectional electric field formed between the first conductive film 201 and the second conductive film 202, opposite to the polarity of the first conductive film 201 on the upper surface of the microfluidic chamber 2, accelerates the sedimentation of cells in the liquid-based cell sample injected into the microfluidic chamber 2.
EXAMPLE III
A third embodiment of the present invention provides a microscopic imaging apparatus for liquid-based cell samples, as shown in fig. 16, comprising:
the photosensitive area 1 of the image acquisition chip is used for acquiring image information of a liquid-based cell sample;
the two micro-flow chambers 2 are arranged on the photosensitive area 1, the lower surface of each micro-flow chamber 2 is attached to the upper surface of the photosensitive area 1, the lower surfaces of the micro-flow chambers 2 are transparent, and the micro-flow chambers 2 are used for loading liquid-based cell samples;
each microfluidic chamber 2 comprises:
a hollow cavity 20;
the two through holes 21 are symmetrically arranged on two sides of the microfluidic chamber 2 and are respectively communicated with two ends of the hollow cavity 20, and the two through holes 21 are higher than the lower surface of the microfluidic chamber 2 and are respectively used for inputting and outputting liquid-based cell samples;
the upper surface of the object carrying platform 3 is provided with a concave part 30, and the photosensitive area 1 of the image acquisition chip is arranged in the concave part 30;
one surface of the micro-flow chamber 2 facing the light-sensing area 1 of the image acquisition chip is provided with a protruding part 22, the bottom of the protruding part 22 is matched with the size of the recessed part 30, so that the protruding part 22 is embedded in the recessed part 30, and the lower surface of the protruding part 22 is attached to the light-sensing area 1 of the image acquisition chip;
the two microfluidic chambers 2 are arranged in parallel in the recess 30 of the stage 3.
Specifically, as shown in fig. 16, two microfluidic chambers 2 are arranged in parallel, and a light-sensitive area 1 of one image acquisition chip can be used to simultaneously detect liquid-based cell samples in the two microfluidic chambers 2; the bottom of the concave part 30 can also be provided with the photosensitive areas 1 of the two image acquisition chips, and the liquid-based cell samples in the two microfluidic chambers 2 can be respectively detected.
It should be noted that, in this embodiment, the two parallel micro-flow chambers 2 are only one preferred embodiment, and a greater number of micro-flow chambers and/or a greater number of light-sensitive areas 1 of the image capturing chip may also be provided according to actual needs, and the number of the micro-flow chambers 2 and the number of the light-sensitive areas 1 of the image capturing chip are not taken as a limitation to the present invention.
Example four
A fourth embodiment of the present invention provides a microscopic imaging method, which is applied to the microscopic imaging apparatus in any of the above embodiments, as shown in fig. 18, and specifically includes 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 microfluidic cavity;
step S2, using the photosensitive area of the image acquisition chip to acquire the image of the diluent in the hollow cavity to form image information;
step S3, the image processor carries out optimization processing on the image information;
in step S4, the optimized image information is displayed on the display.
Specifically, in the present embodiment, as shown in fig. 1, the micro-imaging device includes a photosensitive area 1 and a micro-flow chamber 2, a lower surface of the micro-flow chamber 2 is completely attached to an upper surface of the photosensitive area 1 of the image capturing chip, the lower surface of the micro-flow chamber 2 is matched with the shape and the size of the photosensitive area 1 of the image acquisition chip, the bottom of the micro-flow chamber 2 is in a completely transparent state, so that the liquid-based cell sample at the bottom of the microfluidic chamber 2 can be completely exposed in the collection range of the photosensitive area 1, thereby realizing the microscopic observation and analysis of the full field of view and, at the same time, because the lower surface of the micro-flow chamber 2 is completely attached to the upper surface of the photosensitive area 1, the distance between the lower surface and the photosensitive area is shortened, in order to reduce the influence of light refraction on the microscopic imaging result in the subsequent image acquisition process and effectively improve the quality of the acquired image.
Specifically, the liquid-based cell sample in this embodiment is an exfoliated cell in urine or a diluted liquid of feces or pleural ascites or cerebrospinal fluid or sputum or tracheal mucus of a mammal, or an exfoliated cell in oral cavity or gastric mucosa or a cervical scraping smear of a mammal, or a blood cell or a circulating tumor cell in blood of a mammal. Further, the liquid-based cell sample in this embodiment is preferably human blood. Firstly, taking a proper amount of human blood sample, diluting the human blood, injecting a diluent into the hollow cavity 20 through the through hole 21 on one side, and collecting a microscopic image of the liquid-based cell sample by using the photosensitive area 1 after the diluent fills the bottom of the microfluidic cavity.
As a preferred embodiment, an LED light source may be preset right above the light-sensing area 1 of the image capturing chip, and when taking a bright field photograph, after the diluent fills the bottom of the microfluidic chamber, the LED light source is turned on, and the light-sensing area 1 of the image capturing chip is used to start capturing a microscopic image of the liquid-based cell sample, as shown in fig. 17, a microscopic imaging result of the diluent of the human blood sample is shown, and a microscopic image of a full field of view can be obtained by the above microscopic imaging method, and a large amount of red blood cells are clearly visible.
As a preferred embodiment, the liquid-based cell sample may be stained and then injected into the microfluidic chamber 2. Because the cells are small, colorless and transparent, the contrast between the cells and the background is small, the shapes of the cells are difficult to see, and certain cell structures are difficult to identify, the cells are firstly dyed, the contrast of different parts of the observed cells is improved by means of the contrast effect of the color, and the observation and the research can be carried out more clearly. In addition, certain staining methods can also be used to identify different populations of cells.
As a preferred embodiment, step S1 further includes:
step S11, labeling the diluent with a luciferase gene;
in step S12, the labeled diluent is mixed with a fluorescein substrate.
Specifically, when dark-field photographing is performed, an LED light source is not required to be started, a luciferase gene can be used for marking a liquid-based cell sample before the liquid-based cell sample is injected into the microfluidic chamber, and then the marked liquid-based cell sample and a fluorescein substrate are mixed and then injected into the microfluidic chamber. The entire microfluidic chamber is then enclosed with a light shield to isolate it from ambient light, in a completely dark environment, aimed at imaging the spontaneous fluorescence of the liquid-based cell sample. The light shield material may preferably be an opaque plastic or an opaque metal or other polymer material that is opaque.
In a preferred embodiment, in step S2, before image acquisition, a light shield is used to seal the microfluidic chamber for image acquisition in a dark environment.
Specifically, after the image processor receives a microscopic image formed by autofluorescence of the liquid-based cell sample, noise in the microscopic image is removed or suppressed by an internal preset image processing algorithm, and image data with poor imaging effect is subjected to optimization processing, for example, image data with weak fluorescence signals is subjected to image enhancement processing, image data with blurred edges is subjected to pseudo-color coloring processing, image data with low signal-to-noise ratio is subjected to image segmentation processing, and the like.
The technical scheme of the invention has the beneficial effects that: by adopting the microscopic imaging device in the embodiment of the invention, the lower surface of the microfluidic cavity is attached to the photosensitive area of the image acquisition chip, the distance between the photosensitive area and the liquid-based cell sample is shortened, and the photosensitive area is closer to the liquid-based cell sample, so that the quality of a microscopic image is improved; in addition, because the electrode films with opposite polarities are arranged on the upper surface and the lower surface of the microfluidic chamber, after the liquid-based cell sample is injected into the microfluidic chamber, the unidirectional electric field formed by the two electrode films accelerates the sedimentation speed of the cells so as to solve the technical problem of long sedimentation time caused by natural sedimentation, and the cells can be completely pushed to the bottom of the microfluidic chamber; in addition, the conductive film is made of transparent conductive materials, does not influence the transmission of light, and can be compatible with the existing microscopic imaging device.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

Claims (28)

1. A microscopic imaging apparatus for accelerating cell sedimentation, comprising: the image acquisition chip comprises a photosensitive area and is used for acquiring image information of a liquid-based cell sample; the microfluidic chamber is arranged on the photosensitive area, the lower surface of the microfluidic chamber is attached to the upper surface of the photosensitive area, the lower surface of the microfluidic chamber is transparent, and the microfluidic chamber is used for loading the liquid-based cell sample;
each of the microfluidic chambers includes:
a hollow cavity;
the two through holes are symmetrically arranged on two sides of the micro-flow chamber and are respectively communicated with two ends of the hollow cavity, and the positions of the two through holes are higher than the lower surface of the micro-flow chamber and are respectively used for inputting and outputting the liquid-based cell sample;
the microscopic imaging apparatus further includes:
the two conductive films are respectively covered on the upper surface and the lower surface of the microfluidic chamber, the two conductive films are transparent and have opposite polarities, and the cells in the liquid-based cell sample are accelerated to settle to the bottom of the microfluidic chamber under the action of an electric field formed by the two conductive films.
2. A microscopic imaging apparatus according to claim 1, wherein said microscopic imaging apparatus comprises a plurality of said microfluidic chambers, two of said conductive films comprising:
the first conductive film comprises a plurality of first conductive sub-films, and each first conductive sub-film covers the upper surface of one corresponding micro-flow chamber;
and the second conductive film comprises a plurality of second conductive sub-films, and each second conductive sub-film covers the lower surface of one corresponding micro-flow chamber.
3. The microscopic imaging apparatus according to claim 2, wherein when the liquid-based cell sample injected into the microfluidic chamber is negatively charged, the polarity of the first conductive thin film is negative, and the polarity of the second conductive thin film is positive;
when the liquid-based cell sample injected into the micro-flow chamber is positively charged, the polarity of the first conductive film is positive, and the polarity of the second conductive film is negative.
4. The microscopic imaging apparatus according to claim 2, wherein when the liquid-based cell sample in the microfluidic chamber is negatively charged, the polarity of the corresponding first conductive film is negative, and the polarity of the corresponding second conductive film is positive;
when the liquid-based cell sample in the microfluidic chamber is positively charged, the polarity of the corresponding first conductive film is a positive electrode, and the polarity of the corresponding second conductive film is a negative electrode.
5. A microscopic imaging apparatus according to claim 1, wherein said conductive thin film is made of tin-doped indium oxide or aluminum-doped zinc oxide.
6. A microscopic imaging apparatus according to claim 1, comprising a stage having a recessed portion on an upper surface thereof, wherein said photosensitive region is disposed in said recessed portion.
7. The microscopic imaging apparatus according to claim 6, wherein a surface of the microfluidic chamber facing the photosensitive region has a protrusion, and a bottom of the protrusion is adapted to a size of the recess, so that the protrusion is embedded in the recess, and a lower surface of the protrusion is attached to the photosensitive region.
8. A microscopic imaging apparatus according to claim 7, wherein said protrusion is an inverted trapezoid.
9. A microscopic imaging apparatus according to claim 7, wherein two of said through holes are respectively higher than an upper surface of said protruding portion.
10. The microscopic imaging apparatus according to claim 1, wherein the hollow cavity has a thickness of 50 to 200 μm.
11. The microscopic imaging apparatus according to claim 1, wherein the microfluidic chamber is transparent throughout.
12. A microscopic imaging apparatus according to claim 7, wherein the angle of the apex angle of said protrusion is 90-180 °.
13. A microscopic imaging apparatus according to claim 7, wherein a thickness of said protruding portion is not less than 200 μm.
14. The microscopic imaging apparatus according to claim 1, wherein the hollow cavity has a shape that is oval or circular or rectangular or rounded parallelogram.
15. A microscopic imaging apparatus according to claim 1, wherein said microfluidic chamber is made of transparent glass or transparent organic polymer.
16. A microscopic imaging apparatus according to claim 1, wherein said liquid-based cell sample is labeled with a luciferase gene, and then mixed with a luciferin substrate before entering said microfluidic chamber.
17. A microscopic imaging apparatus according to claim 1, wherein an LED light source is provided above said microscopic imaging apparatus.
18. The microscopic imaging apparatus according to claim 17, further comprising a light shield adapted to fit over said LED light source and sized to fit over said microfluidic chamber such that said light shield is capable of enclosing said microfluidic chamber.
19. The microscopic imaging apparatus according to claim 6, wherein the photosensitive area of the image capturing chip comprises a detector array, the detector array comprises a plurality of detector units, and the number of the detector units is not less than 1 million.
20. A microscopic imaging apparatus according to claim 19, wherein each of said detector units has a size not larger than 1 μm.
21. The microscopic imaging apparatus according to claim 6, wherein the light sensing area of the image capturing chip comprises a plurality of pins, and the pins are respectively led out from two sides of the recess.
22. A microscopic imaging apparatus according to claim 21, further comprising an image processor, wherein said photosensitive region is connected to an input of said image processor through a plurality of said pins, said image processor being configured to process said acquired image information.
23. A microscopic imaging apparatus according to claim 22, wherein said microscopic imaging apparatus further comprises a display, an input of said display being connected to an output of said image processor for displaying said processed image information.
24. The microscopic imaging apparatus according to claim 6, wherein said microscopic imaging apparatus comprises two of said microfluidic chambers, said two microfluidic chambers being juxtaposed in said recess of said stage.
25. A microscopic imaging method, characterized in that it is applied to a microscopic imaging apparatus according to any one of claims 1 to 24, comprising in particular 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 microfluidic chamber;
step S2, using the photosensitive region to perform image acquisition on the diluent in the hollow cavity to form the image information;
step S3, the image processor performs optimization processing on the image information;
and step S4, displaying the optimized image information through a display.
26. A microscopic imaging method according to claim 25, wherein said step S1 further comprises:
step S11, labeling the diluted solution with a luciferase gene;
and step S12, mixing the marked diluent with a fluorescein substrate.
27. The microscopic imaging method according to claim 25, wherein in step S2, before the image acquisition, the microfluidic chamber is sealed with a light shield to perform the image acquisition in a dark environment.
28. A microscopic imaging method according to claim 25, wherein said optimization process comprises an image enhancement process and/or a pseudo color shading process and/or an image segmentation process.
CN202011070001.5A 2020-09-30 2020-09-30 Microscopic imaging device and microscopic imaging method for accelerating cell sedimentation Pending CN112161962A (en)

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