WO2022036807A1 - Living cell culture and real-time observation system and method - Google Patents

Living cell culture and real-time observation system and method Download PDF

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Publication number
WO2022036807A1
WO2022036807A1 PCT/CN2020/117859 CN2020117859W WO2022036807A1 WO 2022036807 A1 WO2022036807 A1 WO 2022036807A1 CN 2020117859 W CN2020117859 W CN 2020117859W WO 2022036807 A1 WO2022036807 A1 WO 2022036807A1
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cell culture
image sensor
sensor chip
real
light source
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PCT/CN2020/117859
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French (fr)
Chinese (zh)
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杨程
沈心雨
闫锋
曹雪芸
林岚昆
孟云龙
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南京大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/10—Petri dish
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/48—Holding appliances; Racks; Supports
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M31/00—Means for providing, directing, scattering or concentrating light
    • C12M31/10—Means for providing, directing, scattering or concentrating light by light emitting elements located inside the reactor, e.g. LED or OLED
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/06—Means for regulation, monitoring, measurement or control, e.g. flow regulation of illumination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/34—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
    • 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
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation

Definitions

  • the invention relates to the technical field of cell culture and microscopic imaging, in particular to a method for culturing living cells and a real-time observation system.
  • the dynamic observation of living cells is widely demanded in the field of life science research and in the medical field.
  • the research on the basic laws of cell life activities is an important basis for all life sciences.
  • By studying the phenomena of life activities at the cellular level more accurate and comprehensive information reflecting the physiological state and process of cells can be obtained, such as the process of cell growth, division, differentiation and death.
  • the interaction and information transmission between cells enables people to better understand some special cell functions in the intercellular population, and to deeply understand the deeper information such as individual differences of cells, such as cancer cell reproduction and metastasis, cell phagocytosis and other processes.
  • the present invention provides a live cell culture and real-time observation system, which specifically includes:
  • a cell culture dish for carrying live cell samples and culture medium for carrying live cell samples and culture medium
  • an image sensor chip the front surface of the image sensor chip is attached to the bottom surface of the cell culture dish, a photosensitive area is formed on the front surface of the image sensor chip, and an opening exposing the photosensitive area is formed at the bottom of the cell culture dish,
  • the image sensor chip is used to record the projection microscopic image of the living cell sample;
  • the light source device fixed above the image sensor chip, the light source device is used to provide a light source for imaging the living cell sample;
  • a live cell incubator used for placing the cell culture dish, the image sensor chip and the light source device, and providing a growth and reproduction environment for the live cell sample;
  • control device connected to the image sensor chip and the light source device respectively, for controlling the image sensor chip and the light source device, and receiving, processing and displaying the projection microscopic image of the living cell sample.
  • the cell culture dish includes a bottom plate provided with the opening and a side wall with the bottom enclosing the bottom plate, and the thickness of the bottom plate is ⁇ 500 ⁇ m, and the thickness of the side wall is ⁇ 500 ⁇ m.
  • the cell culture dish is made of transparent glass or transparent organic polymer.
  • the bottom surface of the cell culture dish is pasted and fixed on the front surface of the image sensor chip by AB glue or UV glue to form a closed shallow groove, so as to carry the living cell sample and the culture solution.
  • the image sensor chip is packaged with a flexible printed circuit board, and the package connection gold wires around the photosensitive area are fixed with chip encapsulation glue.
  • the flexible printed circuit board is externally connected to a connector male seat through a flexible flat cable
  • the control device is provided with a connector female seat adapted to the connector male seat
  • the image The sensor chip is connected with the control device through the flexible flat cable, the connector male seat and the connector female seat.
  • the base material of the flexible flat cable is a polyimide or polyester film with an electronic shielding film on both sides, the shape is a long strip, and the thickness is less than or equal to 100 ⁇ m.
  • the connector male seat is arranged on the back of a connector base plate, and the base material of the connector base plate is a PCB soft and hard composite board, and the front surface is reinforced with a steel sheet.
  • the base material of the connector male seat is metal
  • the connector male seat is provided with a long rectangular groove, and two rows of connector pins are evenly distributed on both sides of the long rectangular groove.
  • the image sensor chip adopts a semi-floating gate transistor or a composite dielectric gate photosensitive detector as the photosensitive pixel unit.
  • the size of a single photosensitive pixel unit of the image sensor chip is ⁇ 500 mm ⁇ 500 nm, and the number of the photosensitive pixel units of the image sensor chip is ⁇ 400 million.
  • the light source device includes:
  • a light source mainboard which integrates a red, green and blue three-color LED light source
  • adjustable brackets are respectively fixed on the side of the light source main board facing the red, green and blue LED light sources, and each adjustable bracket is used to support and fix the light source main board and adjust the illumination of the light source main board high.
  • the red, green and blue LED light sources are connected to the control device through a connecting cable, so as to control the working mode and illumination brightness of the red, green and blue LED light sources through the control device.
  • an insulation layer is provided on the outside of the living cell incubator.
  • a tray is provided inside the living cell incubator for placing the cell culture dish, the image sensor chip and the light source device.
  • a temperature controller and a gas controller are provided inside the living cell incubator, and the temperature and gas concentration in the living cell incubator are regulated by the temperature controller and the gas controller, so that the Cell samples provide a growth and reproduction environment.
  • control device is arranged outside the living cell incubator, and the back of the living cell incubator is provided with a small through hole, which is used for the control device to communicate with the image sensor chip and the The connection wiring of the light source device is described.
  • a live cell culture and real-time observation method applied to the live cell culture and real-time observation system described in any one of the above, the live cell culture and real-time observation method comprising the following steps:
  • Step S1 inoculating live cells and culture liquid to be cultured and observed on the photosensitive area of the image sensor chip in the cell culture dish;
  • Step S2 setting the culture environment for the living cell incubator, and placing the image sensor chip and the cell culture dish inoculated with the living cells and the culture medium, and the light source device on the in the living cell incubator, and connected to the control device;
  • Step S3 the control device controls the light source device to turn on, and simultaneously controls the image sensor chip to collect the projection microscopic image of the living cell sample;
  • step S4 the control device receives the projection microscopic image obtained after collection, and processes the projection microscopic image for display.
  • step S1 before performing the step S1, it also includes:
  • the treatment methods of the sterilization treatment include irradiation with ultraviolet light and soaking with anhydrous alcohol.
  • control device controlling the image sensor chip to collect the projection microscopic image of the living cell sample includes:
  • the control device controls the image sensor chip to acquire the projection microscopic image in an image acquisition mode, so as to observe the current state of the living cell sample;
  • the control device controls the image sensor chip to collect a plurality of the projection microscopic images at a preset video collection time in a video collection mode, so as to perform cell changes of the living cell sample at the preset video collection time. observation.
  • a temperature controller and a gas controller are provided in the living cell incubator, and in step S2, a culture environment is set for the living cell incubator through the temperature controller and the gas controller.
  • the structure is simple and the operation is simple, which greatly improves the portability and convenience of live cell culture and observation, greatly improves the efficiency of live cell culture and observation, reduces the cost of live cell culture and observation, and has a wide range of applications. prospect.
  • FIG. 1 is a schematic structural diagram of a live cell culture and real-time observation system in an embodiment of the present invention
  • Fig. 2 is in the embodiment of the present invention, the principle block diagram of a kind of living cell culture and real-time observation system
  • FIG. 3 is a schematic structural diagram of a cell culture dish in an embodiment of the present invention.
  • FIG. 4 is a schematic structural diagram of a cell culture dish in an embodiment of the present invention.
  • FIG. 5 is a schematic structural diagram of an image sensor chip combined with a cell culture dish in an embodiment of the present invention
  • FIG. 6 is a schematic structural diagram of an image sensor chip in an embodiment of the present invention.
  • FIG. 7 is a schematic structural diagram of a cell imaging device according to an embodiment of the present invention.
  • FIG. 8 is a detailed enlarged schematic diagram of a full-field projection microscopic image of the adherent growth of living cells in an embodiment of the present invention.
  • FIG. 9 is a schematic flowchart of a method for culturing and real-time observation of living cells in an embodiment of the present invention.
  • An image sensor chip 2 the front of the image sensor chip 2 is attached to the bottom surface of the cell culture dish 1, the image sensor chip 2 is provided with a photosensitive area 21 on the front, and the bottom of the cell culture dish 1 is provided with an opening to expose the photosensitive area 21, the image sensor chip 2 for recording projection microscopy images of live cell samples;
  • a light source device 3 is fixed above the image sensor chip 2, and the light source device 3 is used to provide a light source for imaging of living cell samples;
  • a living cell incubator 4 used for placing the cell culture dish 1, the image sensor chip 2 and the light source device 3, and providing a growth and reproduction environment for the living cell samples;
  • a control device 5 respectively connected to the image sensor chip 2 and the light source device 3, is used to control the image sensor chip 2 and the light source device 3, and to receive, process and display the projection microscopic image of the living cell sample.
  • the cell culture dish 1 , the image sensor chip 2 and the light source device 3 together constitute the cell imaging device 6 , wherein the cell culture dish 1 is provided with an opening at the bottom, and the size of the opening is larger than The size of the photosensitive area 21 of the image sensor chip 2, when the bottom surface of the cell culture dish 1 is attached to the front surface of the image sensor chip 2, the photosensitive area 21 can be completely exposed at the bottom of the cell culture dish 1, so that the cell culture dish 1 is used as a living device.
  • the living cell sample can be directly inoculated in the photosensitive area 21, so that the image sensor chip 2 can directly record the projection microscopic image of the living cell sample, which provides a large field of view for the observation of living cells and effectively reduces the observation. cost. Further, by fixing the light source device 3 above the image sensor chip 2, sufficient light source can be provided for imaging the living cell sample while the image sensor chip 2 is performing projection microscopy image acquisition. When culturing and observing live cell samples, the above-mentioned cell imaging device 6 is placed in the live cell incubator 4 as a whole.
  • the sensor chip 2 and the light source device 3 are connected to the control device 5, and the light source device 3 can be controlled to be turned on and off and the illumination brightness can be adjusted according to the needs through the control device 5.
  • the image sensor chip 2 can be controlled by the control device 5 to perform projection microscopy. Image acquisition, and receive the acquired projection microscopic images for processing and display, so as to realize the cultivation and observation of living cell samples.
  • the cell culture dish 1 is composed of a bottom plate 11 with an opening at the bottom and a smooth side wall 12 , wherein the thickness of the bottom plate 11 is less than or equal to 500 ⁇ m, and the thickness of the side wall 12 is less than or equal to 500 ⁇ m.
  • the cell culture dish 1 can be made of transparent glass or transparent organic polymers, which are completely transparent.
  • the above organic polymers include but are not limited to PDMS (polydimethylsiloxane), PMMA (polymethyl methacrylate). ester), PC (polycarbonate), as well as hydrogels, epoxy resins, etc.
  • the shape of the bottom of the cell culture dish 1 may be circular, as shown in FIG. 3, or rectangular, as shown in FIG. 4, but is not limited to these shapes.
  • the bottom surface of the cell culture dish 1 is pasted and fixed on the front surface of the image sensor chip 2 by AB glue or UV glue to form a closed shallow groove, so as to carry the living cell sample and the culture medium.
  • AB glue is obtained by mixing acrylic modified epoxy and modified amine at 1:2, and is pasted between the front of the image sensor chip 2 and the cell culture dish 1 by AB glue or UV glue, so that the two are completely attached to form a joint formation.
  • the shallow groove is closed, and the photosensitive area 21 of the image sensor chip 2 is completely exposed on the bottom surface of the cell culture dish 1 , as shown in FIG.
  • the image sensor chip 2 is packaged with a flexible printed circuit board, and because it is applied to the culture and observation of liquid cell samples, the image sensor chip 2 is located around the photosensitive area 21 of the image sensor chip 2 .
  • the gold wire used for the packaging connection is fixed by chip packaging glue 22, which protects and has the effect of waterproofing.
  • the chip encapsulating adhesive 22 may be UV adhesive or AB adhesive.
  • a bump with a height of about 0.5-1 mm will be formed around it, which effectively protects the image sensor chip 2 from working normally.
  • the flexible printed circuit board is connected to a connector male seat through a flexible flat cable 23, and the control device 5 is provided with a connector female seat adapted to the connector male seat, and the image sensor chip 2 The connection with the control device 5 is established through the flexible flat cable 23 , the connector male seat and the connector female seat.
  • the base material of the flexible flexible cable 23 is polyimide or polyester film, double-sided with electronic shielding film, the shape is a long strip, the length is ⁇ 10cm, the thickness is ⁇ 100 ⁇ m, and has good bendability.
  • the connector male seat is arranged on the back of a connector base plate 24.
  • the base material of the connector base plate 24 is a PCB soft and hard composite board and the front side is reinforced with a steel sheet to enhance the mechanical strength of the connector male seat.
  • the connector male seat is connected with the connector female seat of the control device 5, and the information collected by the image sensor chip 2 is converted into an electrical signal and transmitted to the control device 5 to realize the transmission of the image signal.
  • the base material of the connector male seat is metal, preferably copper alloy, and the connector male seat is provided with a long rectangular groove 25, and two rows of connector pins 26 are evenly distributed on both sides of the long rectangular groove 25, The total number of connector pins 26 is not less than 40, and signal transmission is realized by connecting the connector pins 26 with the connector female seat on the control device 5 .
  • the structure adopted by a single photosensitive pixel unit in the photosensitive region 21 of the image sensor chip 2 can be the composite dielectric grating photosensitive detector described in US Pat. No. 8,604,409, or can be a document (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.) Half floating gate transistor.
  • the size of a single photosensitive pixel unit is ⁇ 500mm ⁇ 500nm, and the number of photosensitive pixel units of the image sensor chip 2 is ⁇ 400 million. In this way, the smaller the pixel size, the higher the resolution, and the finer details of the sample can be seen.
  • the pixel scale of more than 100 million levels ensures a large field of view with high resolution.
  • the light source device 3 includes:
  • the light source main board 31, a red, green and blue three-color LED light source 32 is integrated on the light source main board 31;
  • adjustable brackets 33 are respectively fixed on the side of the light source main board 31 facing the red, green and blue LED light sources 32 .
  • the packaged image sensor chip 2 is disposed on a chip base plate 27, the photosensitive area 21 of the image sensor chip 2 is located at the center of the chip base plate 27, and the bottom surface of the cell culture dish 1 is pasted on the chip base plate 27, And the photosensitive region 21 is completely exposed at the opening on the bottom surface of the cell culture dish 1 .
  • the edge of the chip base plate 27 is provided with a plurality of screw holes 28 , and the adjustable brackets 33 are fixed on the chip base plate 27 through the screw holes 28 to fix the light source device 3 above the image sensor chip 2 .
  • the above-mentioned light source main board 31 can be a square main board, the substrate of the square main board is a PCB soft and hard composite board, and a red, green and blue LED light source 32 is integrated at the geometric center of the square main board, and the red, green and blue LED light source 32 is integrated.
  • 32 is connected to the control device 5 through a connecting cable, so as to control the working mode and illumination brightness of the red, green and blue LED light source 32 through the control device 5 .
  • the complete cell imaging device 6 is connected to the control device 5, which can realize real-time observation of the images of cells cultured at the bottom of the cell culture dish 1 and the upper part of the photosensitive area 21.
  • the material is plastic, and the height can be adjusted.
  • the adjustable structure is a conventional mechanical structure, which is not the focus of the present invention, and will not be repeated here.
  • the living cell incubator 4 can be a box with a total size of 50cm ⁇ 50cm ⁇ 50cm, and a temperature controller and a gas controller 41 are provided in the middle of the living cell incubator 4 for temperature setting. The temperature and gas concentration in the living cell incubator 4 are adjusted through the temperature controller and the gas controller 41 to provide a growth and reproduction environment for the living cell sample.
  • the outside of the box body is provided with a thermal insulation layer with a thickness of 5 cm. Used to maintain constant temperature and gas inside the box.
  • An iron tray 42 is provided inside the living cell incubator 4 for fixing the cell imaging device 6 .
  • the front of the box is provided with a glass door and a shading curtain. The glass door is used to observe the growth of cells inside the box, and the shading curtain is used to control the light conditions for cell growth.
  • control device 5 is arranged outside the living cell incubator 4 , and the back of the living cell incubator 4 is provided with small through holes for the connector male seat, the soft flexible cable 23 and the image sensor chip 2 .
  • the connection cables of the light source device 3 pass through and are connected to the external control device 5 , and silica gel is used for gas tightness at the small through hole.
  • the connector female seat of the control device 5 is connected with the connector male seat, which is used to control the photosensitive area 21 of the image sensor chip 2 and the working mode of the light source device 3, and process the image or video data transmitted from the cell imaging device 6, and display it. As a result, as shown in Fig.
  • the real-time dynamic monitoring of the growth state of living cells can be realized in a full field of view without contacting and moving the sample.
  • This system provides a large field of view while satisfying high resolution, and can monitor tens of thousands or even hundreds of thousands of cell samples at the same time, and can see the relative changes of different cell samples at the same time and at long distances.
  • the continuous culture and real-time observation of live cell samples are realized, which greatly improves the efficiency of live cell culture observation.
  • the system has a simple structure and simple operation, which greatly improves the portability and convenience of live cell culture and observation, and has broad application prospects.
  • the present invention also provides a live cell culture and real-time observation method, which is applied to any of the above-mentioned live cell culture and real-time observation systems.
  • the live cell culture and real-time observation method includes the following steps:
  • Step S1 inoculating the live cells and culture solution to be cultured and observed on the photosensitive area of the image sensor chip in the cell culture dish;
  • Step S2 setting the culture environment for the living cell incubator, placing the image sensor chip and the cell culture dish inoculated with the living cells and the culture solution, and the light source device in the living cell incubator, and connecting to the control device;
  • Step S3 the control device controls the light source device to turn on, and simultaneously controls the image sensor chip to collect the projection microscopic image of the living cell sample;
  • step S4 the control device receives the projection microscopic image obtained by collection after the collection, and processes the projection microscopic image and displays it.
  • taking 091214 primary glioma cells as a live cell sample for live cell culture and real-time observation first add 5 ml of culture medium 90% DMED + 10% FBS to the cell culture dish, and take 091214 original The glioma cells were inoculated into the culture medium as live cell samples.
  • the ambient temperature in the live cell incubator was set to 37°C by the temperature controller in the live cell incubator, and the gas in the live cell incubator was set to 5% CO2, 95% O2 by the gas controller, and in the live cell incubator
  • fix the image sensor chip, cell culture dish, and light source device in the live cell incubator connect the connector male seat and soft cable of the image sensor chip, and the light source device.
  • Connect the cable through the small through hole reserved on the live cell incubator connect it to the external control device, close the door of the live cell incubator, and keep the incubator airtight.
  • the 091214 primary glioma cells were then continuously cultured in a live cell incubator.
  • the control device may be provided with a start acquisition button.
  • the control device When the live cell sample in the cell culture dish needs to be observed, click the start acquisition button, the control device will give a acquisition signal, and control the light source device to turn on, and then click the start acquisition button.
  • the control device controls the light source device to turn off, and reads the projection microscopic image collected by the image sensor chip, and displays the projection microscopic image after processing for Observers to check.
  • step S1 before performing step S1, it also includes:
  • the sterilization treatment method includes irradiating with ultraviolet light for 30 minutes, then soaking in anhydrous alcohol for 30 minutes, then standing for 10 minutes until the surface is completely dry, and then adding a wall to the bottom of the cell culture dish to promote adhesion After 30 minutes of treatment, the liquid was discarded and washed three times with PBS.
  • the above-mentioned adhesion promoting agent may be L-polylysine.
  • control device controlling the image sensor chip to collect the projection microscopic image of the living cell sample includes:
  • the control device controls the image sensor chip to acquire the projection microscopic image in the image acquisition mode, so as to observe the current state of the living cell sample;
  • the control device controls the image sensor chip to collect several projection microscopic images at the preset video collection time in the video collection mode, so as to observe the cell changes of the living cell sample at the preset video collection time.

Abstract

A living cell culture and real-time observation system, relating to the technical fields of cell culture and microscopic imaging. The living cell culture and real-time observation system comprises a cell imaging apparatus. The cell imaging apparatus comprises a cell culture dish (1) for bearing a living cell sample and a culture solution; an image sensor chip (2), which is provided with a photosensitive area (21), wherein the front surface of the image sensor chip (2) is attached to the bottom surface of the cell culture dish (1), and the photosensitive area (21) is completely exposed at the bottom surface of the cell culture dish (1) and is used for recording a projected microscopic image of the living cell sample; and a light source apparatus (3), which is fixed above the image sensor chip (2) and is used for providing a light source for imaging of the living cell sample. The living cell culture and real-time observation system further comprises: a living cell incubator (4) for the cell imaging apparatus to be placed therein and for providing a growth and reproduction environment for the living cell sample; and a control apparatus (5) for controlling the image sensor chip (2) and the light source apparatus (3) and receiving, processing and displaying the projected microscopic image of the living cell sample. By means of the present invention, a large field of view is obtained, the efficiency is improved, phenomena that cannot be observed with a traditional microscope can be observed, and the cost of living cell culture and observation is reduced.

Description

一种活细胞培养和实时观测系统及方法A live cell culture and real-time observation system and method 技术领域technical field
本发明涉及细胞培养和显微成像技术领域，尤其涉及一种活细胞培养和实时观测系统方法。The invention relates to the technical field of cell culture and microscopic imaging, in particular to a method for culturing living cells and a real-time observation system.
背景技术Background technique
活细胞生物动态观测在生命科学研究领域以及医学领域中需求广泛，对细胞生命活动的基本规律研究是一切生命科学的重要基础。在细胞水平上进行生命活动现象的研究，可以获得更准确、更全面的反映细胞生理状态和过程的信息，例如细胞生长、分裂、分化和死亡的过程。而细胞间的相互作用和信息传递，使人们更好地了解细胞间群体中某些特殊的细胞功能，深入认识细胞个体差异等更深层次的信息，例如癌细胞繁殖转移、细胞吞噬等过程。由于细胞形态发生变化的速度快，一些生理行为特征周期时间短，因而活细胞观测分析手段要满足实时、响应速度快、连续、高灵敏、高时空分辨率等要求，同时对生物样品还需要满足无损或微损的要求。The dynamic observation of living cells is widely demanded in the field of life science research and in the medical field. The research on the basic laws of cell life activities is an important basis for all life sciences. By studying the phenomena of life activities at the cellular level, more accurate and comprehensive information reflecting the physiological state and process of cells can be obtained, such as the process of cell growth, division, differentiation and death. The interaction and information transmission between cells enables people to better understand some special cell functions in the intercellular population, and to deeply understand the deeper information such as individual differences of cells, such as cancer cell reproduction and metastasis, cell phagocytosis and other processes. Due to the rapid change of cell morphology and the short cycle time of some physiological behavior characteristics, the observation and analysis methods of living cells must meet the requirements of real-time, fast response, continuous, high sensitivity, and high temporal and spatial resolution. Non-destructive or minimal loss requirements.
当前主要的细胞培养和监测过程主要存在两个问题。第一，整个过程均为人工且重复性的操作，存在效率低下、易受污染、细胞样本发生人为移动和变化等问题。第二，目前细胞生物动态观测都是在显微镜下进行，而在高倍显微镜下的观测一直存在视场小，观测范围受限，无法实现同时观测远距离的不同样本变化，这也是生物医学界目 前尚未解决的一大难题。且因显微镜设备体积大，价格昂贵等问题，细胞相关研究无法广泛普及。There are two main problems with the current major cell culture and monitoring processes. First, the entire process is a manual and repetitive operation, which has problems such as low efficiency, easy contamination, and artificial movement and changes in cell samples. Second, the current cell biological dynamic observation is carried out under the microscope, and the observation under the high-power microscope has always had a small field of view and a limited observation range, and it is impossible to observe the changes of different samples at a long distance at the same time. A big unsolved problem. In addition, due to the large size and high price of microscope equipment, cell-related research cannot be widely popularized.
因此，为了满足广泛的医学、药学和生命科学领域的科研和临床需求，大视场、结构简单、效率高、操作简便、成本较低的活细胞培养和实时观测装置便显得尤为重要。Therefore, in order to meet the scientific research and clinical needs in a wide range of medical, pharmaceutical and life science fields, living cell culture and real-time observation devices with large field of view, simple structure, high efficiency, simple operation, and low cost are particularly important.
发明内容SUMMARY OF THE INVENTION
针对现有技术中存在的问题，本发明提供一种活细胞培养和实时观测系统，具体包括：In view of the problems existing in the prior art, the present invention provides a live cell culture and real-time observation system, which specifically includes:
一细胞培养皿，用于承载活细胞样本和培养液；A cell culture dish for carrying live cell samples and culture medium;
一图像传感器芯片，所述图像传感器芯片的正面与所述细胞培养皿的底面贴合，所述图像传感器芯片正面设有一感光区且所述细胞培养皿底部设有暴露所述感光区的开口，所述图像传感器芯片用于记录所述活细胞样本的投影显微图像；an image sensor chip, the front surface of the image sensor chip is attached to the bottom surface of the cell culture dish, a photosensitive area is formed on the front surface of the image sensor chip, and an opening exposing the photosensitive area is formed at the bottom of the cell culture dish, The image sensor chip is used to record the projection microscopic image of the living cell sample;
一光源装置，固定于所述图像传感器芯片上方，所述光源装置用于为所述活细胞样本成像提供光源；a light source device fixed above the image sensor chip, the light source device is used to provide a light source for imaging the living cell sample;
一活细胞培养箱，用于放置所述细胞培养皿、所述图像传感器芯片和所述光源装置，并为所述活细胞样本提供生长繁殖环境；a live cell incubator, used for placing the cell culture dish, the image sensor chip and the light source device, and providing a growth and reproduction environment for the live cell sample;
一控制装置，分别连接所述图像传感器芯片和所述光源装置，用于控制所述图像传感器芯片和所述光源装置，并接收、处理和显示所述活细胞样本的所述投影显微图像。a control device, connected to the image sensor chip and the light source device respectively, for controlling the image sensor chip and the light source device, and receiving, processing and displaying the projection microscopic image of the living cell sample.
优选的，所述细胞培养皿包括设有所述开口的底板以及底部围合 所述底板的侧壁，且所述底板的厚度≤500μm，所述侧壁的厚度≤500μm。Preferably, the cell culture dish includes a bottom plate provided with the opening and a side wall with the bottom enclosing the bottom plate, and the thickness of the bottom plate is ≤ 500 μm, and the thickness of the side wall is ≤ 500 μm.
优选的，所述细胞培养皿的制作材料为透明玻璃，或透明的有机聚合物。Preferably, the cell culture dish is made of transparent glass or transparent organic polymer.
优选的，所述细胞培养皿的底面通过AB胶或紫外胶粘贴固定在所述图像传感器芯片的正面形成封闭浅槽，以承载所述活细胞样本和所述培养液。Preferably, the bottom surface of the cell culture dish is pasted and fixed on the front surface of the image sensor chip by AB glue or UV glue to form a closed shallow groove, so as to carry the living cell sample and the culture solution.
优选的，所述图像传感器芯片采用柔性印制电路板封装，且所述感光区周围的封装连接用金线采用芯片封装胶固定。Preferably, the image sensor chip is packaged with a flexible printed circuit board, and the package connection gold wires around the photosensitive area are fixed with chip encapsulation glue.
优选的，所述柔性印制电路板通过一软质排线外接一连接器公座，且所述控制装置上设有与所述连接器公座适配的一连接器母座，所述图像传感器芯片通过所述软质排线、所述连接器公座和所述连接器母座与所述控制装置建立连接。Preferably, the flexible printed circuit board is externally connected to a connector male seat through a flexible flat cable, and the control device is provided with a connector female seat adapted to the connector male seat, and the image The sensor chip is connected with the control device through the flexible flat cable, the connector male seat and the connector female seat.
优选的，所述软质排线的基材为以聚酰亚胺或聚酯薄膜，双面加电子屏蔽膜，形状为长条型，厚度≤100μm。Preferably, the base material of the flexible flat cable is a polyimide or polyester film with an electronic shielding film on both sides, the shape is a long strip, and the thickness is less than or equal to 100 μm.
优选的，所述连接器公座设置于一连接器底板的背面，所述连接器底板的基材为PCB软硬复合板且正面加钢片补强。Preferably, the connector male seat is arranged on the back of a connector base plate, and the base material of the connector base plate is a PCB soft and hard composite board, and the front surface is reinforced with a steel sheet.
优选的，所述连接器公座的基材为金属，且所述连接器公座上设有一长矩形沟槽，所述长矩形沟槽两侧均匀分布有两排连接器引脚。Preferably, the base material of the connector male seat is metal, and the connector male seat is provided with a long rectangular groove, and two rows of connector pins are evenly distributed on both sides of the long rectangular groove.
优选的，所述图像传感器芯片采用半浮栅晶体管或复合介质栅光敏探测器作为感光像素单元。Preferably, the image sensor chip adopts a semi-floating gate transistor or a composite dielectric gate photosensitive detector as the photosensitive pixel unit.
优选的，所述图像传感器芯片的单个所述感光像素单元的尺寸≤ 500mm×500nm，所述图像传感器芯片的所述感光像素单元的数目≥4亿。Preferably, the size of a single photosensitive pixel unit of the image sensor chip is ≤ 500 mm×500 nm, and the number of the photosensitive pixel units of the image sensor chip is ≥ 400 million.
优选的，所述光源装置包括：Preferably, the light source device includes:
光源主板，所述光源主板上集成一红绿蓝三色LED光源；a light source mainboard, which integrates a red, green and blue three-color LED light source;
若干可调节支架，分别固定于所述光源主板的朝向所述红绿蓝三色LED光源的一侧，各所述可调节支架用于支撑和固定所述光源主板并调节所述光源主板的照明高度。Several adjustable brackets are respectively fixed on the side of the light source main board facing the red, green and blue LED light sources, and each adjustable bracket is used to support and fix the light source main board and adjust the illumination of the light source main board high.
优选的，所述红绿蓝三色LED光源通过一连接排线与所述控制装置连接，以通过所述控制装置控制所述红绿蓝三色LED光源的工作模式和照明亮度。Preferably, the red, green and blue LED light sources are connected to the control device through a connecting cable, so as to control the working mode and illumination brightness of the red, green and blue LED light sources through the control device.
优选的，所述活细胞培养箱的外部设有保温层。Preferably, an insulation layer is provided on the outside of the living cell incubator.
优选的，所述活细胞培养箱的内部设有一托盘，用于放置所述细胞培养皿、所述图像传感器芯片和所述光源装置。Preferably, a tray is provided inside the living cell incubator for placing the cell culture dish, the image sensor chip and the light source device.
优选的，所述活细胞培养箱内部设有温度控制器和气体控制器，通过所述温度控制器和所述气体控制器调节所述活细胞培养箱中的温度和气体浓度，为所述活细胞样本提供生长繁殖环境。Preferably, a temperature controller and a gas controller are provided inside the living cell incubator, and the temperature and gas concentration in the living cell incubator are regulated by the temperature controller and the gas controller, so that the Cell samples provide a growth and reproduction environment.
优选的，所述控制装置设置于所述活细胞培养箱的外部，所述活细胞培养箱的箱体背面设有小型通孔，用于供所述控制装置分别与所述图像传感器芯片和所述光源装置的连接走线。Preferably, the control device is arranged outside the living cell incubator, and the back of the living cell incubator is provided with a small through hole, which is used for the control device to communicate with the image sensor chip and the The connection wiring of the light source device is described.
一种活细胞培养和实时观测方法，应用于上述任意一项所述的活细胞培养和实时观测系统，所述活细胞培养和实时观测方法包括以下步骤：A live cell culture and real-time observation method, applied to the live cell culture and real-time observation system described in any one of the above, the live cell culture and real-time observation method comprising the following steps:
步骤S1，将待培养和观测的活细胞和培养液接种至所述细胞培养皿内的所述图像传感器芯片的所述感光区上；Step S1, inoculating live cells and culture liquid to be cultured and observed on the photosensitive area of the image sensor chip in the cell culture dish;
步骤S2，对所述活细胞培养箱进行培养环境设置，并将接种有所述活细胞和所述培养液的所述图像传感器芯片和所述细胞培养皿，以及所述光源装置放置于所述活细胞培养箱内，并接入所述控制装置；Step S2, setting the culture environment for the living cell incubator, and placing the image sensor chip and the cell culture dish inoculated with the living cells and the culture medium, and the light source device on the in the living cell incubator, and connected to the control device;
步骤S3，所述控制装置控制所述光源装置开启，同时控制所述图像传感器芯片对所述活细胞样本的所述投影显微图像进行采集；Step S3, the control device controls the light source device to turn on, and simultaneously controls the image sensor chip to collect the projection microscopic image of the living cell sample;
步骤S4，所述控制装置在采集结束后接收采集得到的所述投影显微图像，并对所述投影显微图像进行处理后显示。In step S4 , the control device receives the projection microscopic image obtained after collection, and processes the projection microscopic image for display.
优选的，执行所述步骤S1之前还包括：Preferably, before performing the step S1, it also includes:
对所述细胞培养皿、所述图像传感器芯片和所述光源装置进行灭菌处理，随后于所述细胞培养皿底部加促贴壁剂进行处理。Sterilize the cell culture dish, the image sensor chip and the light source device, and then add an adhesion promoting agent to the bottom of the cell culture dish for treatment.
优选的，所述灭菌处理的处理方式包括采用紫外光照射和采用无水酒精浸泡。Preferably, the treatment methods of the sterilization treatment include irradiation with ultraviolet light and soaking with anhydrous alcohol.
优选的，所述步骤S3中，所述控制装置控制所述图像传感器芯片对所述活细胞样本的所述投影显微图像进行采集包括：Preferably, in the step S3, the control device controlling the image sensor chip to collect the projection microscopic image of the living cell sample includes:
所述控制装置控制所述图像传感器芯片以图像采集模式采集得到所述投影显微图像，以对所述活细胞样本的当前状态进行观测；或The control device controls the image sensor chip to acquire the projection microscopic image in an image acquisition mode, so as to observe the current state of the living cell sample; or
所述控制装置控制所述图像传感器芯片以视频采集模式采集得到预设视频采集时间的若干所述投影显微图像，以对所述活细胞样本在所述预设视频采集时间的细胞变化情况进行观测。The control device controls the image sensor chip to collect a plurality of the projection microscopic images at a preset video collection time in a video collection mode, so as to perform cell changes of the living cell sample at the preset video collection time. observation.
优选的，所述活细胞培养箱内设有温度控制器和气体控制器，所述步骤S2中，通过所述温度控制器和所述气体控制器对所述活细胞培养箱进行培养环境设置。Preferably, a temperature controller and a gas controller are provided in the living cell incubator, and in step S2, a culture environment is set for the living cell incubator through the temperature controller and the gas controller.
上述技术方案具有如下优点或有益效果：The above-mentioned technical scheme has the following advantages or beneficial effects:
1)能在无接触、不移动样品的前提下，对活细胞进行持续培养并实现活细胞生长状态的全视野实时动态监测；1) Under the premise of no contact and no movement of the sample, it can continuously cultivate living cells and realize real-time dynamic monitoring of the growth state of living cells in a full field of view;
2)在满足高分辨率的同时提供了极大视野，可以实现数万细胞样品的同时监测，可以看到同一时刻下、远距离的不同细胞样本的相对变化；2) It provides a large field of view while satisfying high resolution, which can realize simultaneous monitoring of tens of thousands of cell samples, and can see the relative changes of different cell samples at the same time and long distances;
3)通过将全视野的图像数据保存，可以随时观察全视野中任何一个位置的任何一个细胞变化的全过程；3) By saving the image data of the full field of view, the whole process of the change of any cell at any position in the full field of view can be observed at any time;
4)结构简便，操作简单，极大地提升了活细胞培养和观测的便携性和简便性，极大提高了活细胞培养和观测的效率，降低了活细胞培养和观测的成本，有着广阔的应用前景。4) The structure is simple and the operation is simple, which greatly improves the portability and convenience of live cell culture and observation, greatly improves the efficiency of live cell culture and observation, reduces the cost of live cell culture and observation, and has a wide range of applications. prospect.
附图说明Description of drawings
图1为本发明的实施例中，一种活细胞培养和实时观测系统的结构示意图；1 is a schematic structural diagram of a live cell culture and real-time observation system in an embodiment of the present invention;
图2为本发明的实施例中，一种活细胞培养和实时观测系统的原理框图；Fig. 2 is in the embodiment of the present invention, the principle block diagram of a kind of living cell culture and real-time observation system;
图3为本发明的实施例中，细胞培养皿的结构示意图；3 is a schematic structural diagram of a cell culture dish in an embodiment of the present invention;
图4为本发明的实施例中，细胞培养皿的结构示意图；4 is a schematic structural diagram of a cell culture dish in an embodiment of the present invention;
图5为本发明的实施例中，图像传感器芯片结合细胞培养皿的结构示意图；5 is a schematic structural diagram of an image sensor chip combined with a cell culture dish in an embodiment of the present invention;
图6为本发明的实施例中，图像传感器芯片的结构示意图；6 is a schematic structural diagram of an image sensor chip in an embodiment of the present invention;
图7为本发明的实施例中，细胞成像装置的结构示意图；7 is a schematic structural diagram of a cell imaging device according to an embodiment of the present invention;
图8为本发明的实施例中，活细胞贴壁生长的全视野投影显微图像的细节放大示意图；8 is a detailed enlarged schematic diagram of a full-field projection microscopic image of the adherent growth of living cells in an embodiment of the present invention;
图9为本发明的实施例中，一种活细胞培养和实时观测方法的流程示意图。FIG. 9 is a schematic flowchart of a method for culturing and real-time observation of living cells in an embodiment of the present invention.
具体实施方式detailed description
下面结合附图和具体实施例对本发明进行详细说明。本发明并不限定于该实施方式，只要符合本发明的主旨，则其他实施方式也可以属于本发明的范畴。The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The present invention is not limited to this embodiment, and other embodiments may belong to the scope of the present invention as long as it conforms to the gist of the present invention.
实施例一Example 1
基于现有技术中存在的上述问题，现提供一种活细胞培养和实时观测系统，具体包括：Based on the above problems existing in the prior art, a live cell culture and real-time observation system is now provided, which specifically includes:
一细胞培养皿1，用于承载活细胞样本和培养液；A cell culture dish 1 for carrying live cell samples and culture medium;
一图像传感器芯片2，图像传感器芯片2的正面与细胞培养皿1的底面贴合，图像传感器芯片2正面设有一感光区21且细胞培养皿1底部设有暴露感光区21的开口，图像传感器芯片2用于记录活细胞样本的投影显微图像；An image sensor chip 2, the front of the image sensor chip 2 is attached to the bottom surface of the cell culture dish 1, the image sensor chip 2 is provided with a photosensitive area 21 on the front, and the bottom of the cell culture dish 1 is provided with an opening to expose the photosensitive area 21, the image sensor chip 2 for recording projection microscopy images of live cell samples;
一光源装置3，固定于图像传感器芯片2上方，光源装置3用于 为活细胞样本成像提供光源；A light source device 3 is fixed above the image sensor chip 2, and the light source device 3 is used to provide a light source for imaging of living cell samples;
一活细胞培养箱4，用于放置细胞培养皿1、图像传感器芯片2和光源装置3，并为活细胞样本提供生长繁殖环境；A living cell incubator 4, used for placing the cell culture dish 1, the image sensor chip 2 and the light source device 3, and providing a growth and reproduction environment for the living cell samples;
一控制装置5，分别连接图像传感器芯片2和光源装置3，用于控制图像传感器芯片2和光源装置3，并接收、处理和显示活细胞样本的投影显微图像。A control device 5, respectively connected to the image sensor chip 2 and the light source device 3, is used to control the image sensor chip 2 and the light source device 3, and to receive, process and display the projection microscopic image of the living cell sample.
上述实施例中，如图1至图5所示，细胞培养皿1、图像传感器芯片2和光源装置3共同组成细胞成像装置6，其中，细胞培养皿1底部设有开口，该开口的尺寸大于图像传感器芯片2的感光区21的尺寸，细胞培养皿1的底面与图像传感器芯片2的正面贴合时，感光区21能够完全暴露在细胞培养皿1的底部，使得采用细胞培养皿1作为活细胞样本和培养液的载体的同时，活细胞样本能够直接接种在感光区21，以便图像传感器芯片2直接记录活细胞样本的投影显微图像，为活细胞观测提供大视场的同时有效降低观测成本。进一步地，通过在图像传感器芯片2的上方固定光源装置3，能够在图像传感器芯片2进行投影显微图像采集的同时为活细胞样本成像提供足够的光源。在进行活细胞样本的培养和观测时，将上述细胞成像装置6整体放置于活细胞培养箱4中，该活细胞培养箱4能够为活细胞样本的培养提供适宜的生长繁殖环境，随后将图像传感器芯片2和光源装置3接入控制装置5，通过控制装置5可以控制光源装置3的开启和关闭以及根据需要对照明亮度进行调节，同时通过控制装置5可以控制图像传感器芯片2进行投影显微图像的采集，并接收采集得到的投影 显微图像进行处理后显示，实现活细胞样本的培养和观测。In the above embodiment, as shown in FIGS. 1 to 5 , the cell culture dish 1 , the image sensor chip 2 and the light source device 3 together constitute the cell imaging device 6 , wherein the cell culture dish 1 is provided with an opening at the bottom, and the size of the opening is larger than The size of the photosensitive area 21 of the image sensor chip 2, when the bottom surface of the cell culture dish 1 is attached to the front surface of the image sensor chip 2, the photosensitive area 21 can be completely exposed at the bottom of the cell culture dish 1, so that the cell culture dish 1 is used as a living device. At the same time as the carrier of the cell sample and the culture medium, the living cell sample can be directly inoculated in the photosensitive area 21, so that the image sensor chip 2 can directly record the projection microscopic image of the living cell sample, which provides a large field of view for the observation of living cells and effectively reduces the observation. cost. Further, by fixing the light source device 3 above the image sensor chip 2, sufficient light source can be provided for imaging the living cell sample while the image sensor chip 2 is performing projection microscopy image acquisition. When culturing and observing live cell samples, the above-mentioned cell imaging device 6 is placed in the live cell incubator 4 as a whole. The sensor chip 2 and the light source device 3 are connected to the control device 5, and the light source device 3 can be controlled to be turned on and off and the illumination brightness can be adjusted according to the needs through the control device 5. At the same time, the image sensor chip 2 can be controlled by the control device 5 to perform projection microscopy. Image acquisition, and receive the acquired projection microscopic images for processing and display, so as to realize the cultivation and observation of living cell samples.
作为优选的实施方式，细胞培养皿1由底部镂空设有开口的底板11以及光滑的侧壁12构成，其中，底板11的厚度≤500μm，侧壁12的厚度≤500μm。细胞培养皿1的制作材料可以是透明玻璃，也可以是透明的有机聚合物，完全透明，上述有机聚合物包括但不限于PDMS(聚二甲基硅氧烷)、PMMA(聚甲基丙烯酸甲酯)、PC(聚碳酸酯)以及水凝胶、环氧树脂等。细胞培养皿1的底部形状可以是圆形，如图3所示，也可以是矩形，如图4所示，但不限于这些形状。As a preferred embodiment, the cell culture dish 1 is composed of a bottom plate 11 with an opening at the bottom and a smooth side wall 12 , wherein the thickness of the bottom plate 11 is less than or equal to 500 μm, and the thickness of the side wall 12 is less than or equal to 500 μm. The cell culture dish 1 can be made of transparent glass or transparent organic polymers, which are completely transparent. The above organic polymers include but are not limited to PDMS (polydimethylsiloxane), PMMA (polymethyl methacrylate). ester), PC (polycarbonate), as well as hydrogels, epoxy resins, etc. The shape of the bottom of the cell culture dish 1 may be circular, as shown in FIG. 3, or rectangular, as shown in FIG. 4, but is not limited to these shapes.
作为优选的实施方式，细胞培养皿1的底面通过AB胶或紫外胶粘贴固定在图像传感器芯片2的正面形成封闭浅槽，以承载活细胞样本和培养液。上述AB胶为丙烯酸改性环氧和改性胺1:2混合得到，通过AB胶或紫外胶粘贴在图像传感器芯片2的正面和细胞培养皿1之间使二者完全贴合，共同形成封闭浅槽，此时图像传感器芯片2的感光区21完全裸露于细胞培养皿1的底面，如图5所示，形成图像传感器芯片2结合细胞培养皿1的结构。As a preferred embodiment, the bottom surface of the cell culture dish 1 is pasted and fixed on the front surface of the image sensor chip 2 by AB glue or UV glue to form a closed shallow groove, so as to carry the living cell sample and the culture medium. The above AB glue is obtained by mixing acrylic modified epoxy and modified amine at 1:2, and is pasted between the front of the image sensor chip 2 and the cell culture dish 1 by AB glue or UV glue, so that the two are completely attached to form a joint formation. The shallow groove is closed, and the photosensitive area 21 of the image sensor chip 2 is completely exposed on the bottom surface of the cell culture dish 1 , as shown in FIG.
作为优选的实施方式，如图5至图7所示，图像传感器芯片2采用柔性印制电路板封装，且由于应用于液体细胞样品的培养和观测，因此在图像传感器芯片2的感光区21周围的封装连接用金线采用芯片封装胶22固定，保护的同时起到防水的效果。其中，芯片封装胶22可以是紫外胶，也可以是AB胶，同时，由于芯片封装胶22固化后会在四周形成高度约为0.5-1mm的凸起，有效保护图像传感器芯片2内部正常工作。As a preferred embodiment, as shown in FIGS. 5 to 7 , the image sensor chip 2 is packaged with a flexible printed circuit board, and because it is applied to the culture and observation of liquid cell samples, the image sensor chip 2 is located around the photosensitive area 21 of the image sensor chip 2 . The gold wire used for the packaging connection is fixed by chip packaging glue 22, which protects and has the effect of waterproofing. The chip encapsulating adhesive 22 may be UV adhesive or AB adhesive. At the same time, after the chip encapsulating adhesive 22 is cured, a bump with a height of about 0.5-1 mm will be formed around it, which effectively protects the image sensor chip 2 from working normally.
作为优选的实施方式，柔性印制电路板通过一软质排线23外接一连接器公座，且控制装置5上设有与连接器公座适配的一连接器母座，图像传感器芯片2通过软质排线23、连接器公座和连接器母座与控制装置5建立连接。As a preferred embodiment, the flexible printed circuit board is connected to a connector male seat through a flexible flat cable 23, and the control device 5 is provided with a connector female seat adapted to the connector male seat, and the image sensor chip 2 The connection with the control device 5 is established through the flexible flat cable 23 , the connector male seat and the connector female seat.
进一步地，软质排线23的基材为以聚酰亚胺或聚酯薄膜，双面加电子屏蔽膜，形状为长条型，长度≥10cm，厚度≤100μm，有良好的弯折性。Further, the base material of the flexible flexible cable 23 is polyimide or polyester film, double-sided with electronic shielding film, the shape is a long strip, the length is ≥10cm, the thickness is ≤100μm, and has good bendability.
进一步地，连接器公座设置于一连接器底板24的背面，连接器底板24的基材为PCB软硬复合板且正面加钢片补强，用以增强连接器公座的机械强度，通过连接器公座与控制装置5的连接器母座连接，将图像传感器芯片2采集的信息转化为电信号传递给控制装置5，实现图像信号的传输。Further, the connector male seat is arranged on the back of a connector base plate 24. The base material of the connector base plate 24 is a PCB soft and hard composite board and the front side is reinforced with a steel sheet to enhance the mechanical strength of the connector male seat. The connector male seat is connected with the connector female seat of the control device 5, and the information collected by the image sensor chip 2 is converted into an electrical signal and transmitted to the control device 5 to realize the transmission of the image signal.
进一步地，连接器公座的基材为金属，优选为铜合金，且连接器公座上设有一长矩形沟槽25，长矩形沟槽25两侧均匀分布有两排连接器引脚26，连接器引脚26的总数不小于40，通过该连接器引脚26与控制装置5上的连接器母座相连，实现信号传输。Further, the base material of the connector male seat is metal, preferably copper alloy, and the connector male seat is provided with a long rectangular groove 25, and two rows of connector pins 26 are evenly distributed on both sides of the long rectangular groove 25, The total number of connector pins 26 is not less than 40, and signal transmission is realized by connecting the connector pins 26 with the connector female seat on the control device 5 .
作为优选的实施方式，图像传感器芯片2的感光区21中单个感光像素单元采用的结构可以是美国专利US 8,604,409中所述的复合介质栅光敏探测器，还可以是文献(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,N.Y.),2013,341(6146):640-643.)中所述的半浮栅晶体管。并且单个感光 像素单元的尺寸≤500mm×500nm，图像传感器芯片2的感光像素单元的数目≥4亿。这样像元尺寸越小分辨率越高，能看到越细微的样品细节信息，同时亿级以上的像素规模保证了在高分辨率的情况下兼备大的视场。As a preferred embodiment, the structure adopted by a single photosensitive pixel unit in the photosensitive region 21 of the image sensor chip 2 can be the composite dielectric grating photosensitive detector described in US Pat. No. 8,604,409, or can be a document (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.) Half floating gate transistor. And the size of a single photosensitive pixel unit is ≤500mm×500nm, and the number of photosensitive pixel units of the image sensor chip 2 is ≥400 million. In this way, the smaller the pixel size, the higher the resolution, and the finer details of the sample can be seen. At the same time, the pixel scale of more than 100 million levels ensures a large field of view with high resolution.
作为优选的实施方式，如图7所示，光源装置3包括：As a preferred embodiment, as shown in FIG. 7 , the light source device 3 includes:
光源主板31，光源主板31上集成一红绿蓝三色LED光源32；The light source main board 31, a red, green and blue three-color LED light source 32 is integrated on the light source main board 31;
若干可调节支架33，分别固定于光源主板31的朝向红绿蓝三色LED光源32的一侧，各可调节支架33用于支撑和固定光源主板31并调节光源主板31的照明高度。Several adjustable brackets 33 are respectively fixed on the side of the light source main board 31 facing the red, green and blue LED light sources 32 .
通过对图像传感器芯片2结合细胞培养皿1的结构上增加光源装置3，形成完整的细胞成像装置。作为优选的实施方式，封装后的图像传感器芯片2设置于一芯片底板27上，图像传感器芯片2的感光区21位于芯片底板27的中心位置，细胞培养皿1的底面粘贴于芯片底板27上，且感光区21完全暴露于细胞培养皿1底面的开口处。该芯片底板27的边缘处设有若干螺丝孔28，各可调节支架33通过各螺丝孔28固定于芯片底板27上，以将光源装置3固定于图像传感器芯片2的上方。By adding a light source device 3 to the structure of the image sensor chip 2 combined with the cell culture dish 1, a complete cell imaging device is formed. As a preferred embodiment, the packaged image sensor chip 2 is disposed on a chip base plate 27, the photosensitive area 21 of the image sensor chip 2 is located at the center of the chip base plate 27, and the bottom surface of the cell culture dish 1 is pasted on the chip base plate 27, And the photosensitive region 21 is completely exposed at the opening on the bottom surface of the cell culture dish 1 . The edge of the chip base plate 27 is provided with a plurality of screw holes 28 , and the adjustable brackets 33 are fixed on the chip base plate 27 through the screw holes 28 to fix the light source device 3 above the image sensor chip 2 .
进一步地，上述光源主板31可以是正方形主板，该正方形主板的基板为PCB软硬复合板，且正方形主板的几何中心处集成一个红绿蓝三色LED光源32，该红绿蓝三色LED光源32通过连接排线与控制装置5相连，以通过控制装置5控制红绿蓝三色LED光源32的工作模式和照明亮度。完整的细胞成像装置6与控制装置5连接，可以实 现对细胞培养皿1底部，感光区21上部培养的细胞图像进行实时观测。Further, the above-mentioned light source main board 31 can be a square main board, the substrate of the square main board is a PCB soft and hard composite board, and a red, green and blue LED light source 32 is integrated at the geometric center of the square main board, and the red, green and blue LED light source 32 is integrated. 32 is connected to the control device 5 through a connecting cable, so as to control the working mode and illumination brightness of the red, green and blue LED light source 32 through the control device 5 . The complete cell imaging device 6 is connected to the control device 5, which can realize real-time observation of the images of cells cultured at the bottom of the cell culture dish 1 and the upper part of the photosensitive area 21.
进一步地，可调节支架为4根，材质为塑料，高度可调节，具体地可调节结构为常规机械结构，不作为本发明的重点，此处不再赘述。Further, there are four adjustable brackets, the material is plastic, and the height can be adjusted. Specifically, the adjustable structure is a conventional mechanical structure, which is not the focus of the present invention, and will not be repeated here.
作为优选的实施方式，活细胞培养箱4可以是总尺寸为50cm×50cm×50cm的箱体，该活细胞培养箱4的中间位置设有温度控制器和气体控制器41，用于进行温度设定以及气体设定，通过温度控制器和气体控制器41调节活细胞培养箱4中的温度和气体浓度，为活细胞样本提供生长繁殖环境。As a preferred embodiment, the living cell incubator 4 can be a box with a total size of 50cm×50cm×50cm, and a temperature controller and a gas controller 41 are provided in the middle of the living cell incubator 4 for temperature setting. The temperature and gas concentration in the living cell incubator 4 are adjusted through the temperature controller and the gas controller 41 to provide a growth and reproduction environment for the living cell sample.
进一步地，箱体的外部设有厚度为5cm的保温层。用于维持箱体内的温度和气体恒定。该活细胞培养箱4的内部设有一铁质托盘42，用于固定细胞成像装置6。箱体正面设置玻璃门和遮光门帘，玻璃门用于观察箱体内部的细胞生长情况，遮光门帘用于控制细胞生长的光照条件。Further, the outside of the box body is provided with a thermal insulation layer with a thickness of 5 cm. Used to maintain constant temperature and gas inside the box. An iron tray 42 is provided inside the living cell incubator 4 for fixing the cell imaging device 6 . The front of the box is provided with a glass door and a shading curtain. The glass door is used to observe the growth of cells inside the box, and the shading curtain is used to control the light conditions for cell growth.
进一步地，控制装置5设置于活细胞培养箱4的外部，活细胞培养箱4的箱体背面设有小型通孔，用于图像传感器芯片2上的连接器公座、软质排线23以及光源装置3的连接排线通过，并连接到外部的控制装置5，且在该小型通孔处采用硅胶做好气体密封性。控制装置5的连接器母座与连接器公座相连，用于控制图像传感器芯片2的感光区21和光源装置3的工作模式，并处理细胞成像装置6传输出来的图像或视频数据，并显示结果，如图8所示，能在无接触、不移动样品的前提下，实现活细胞生长状态的全视野实时动态监测。本 系统在满足高分辨率的同时提供了极大视野，可以实现数万甚至十几万个细胞样品的同时监测，可以看到同一时刻下、远距离的不同细胞样本的相对变化。实现了对活细胞样本的持续培养和实时观测，极大提高了活细胞培养观测的效率。系统结构简便，操作简单，极大地提升了活细胞培养和观测的便携性和简便性，有着广阔的应用前景。Further, the control device 5 is arranged outside the living cell incubator 4 , and the back of the living cell incubator 4 is provided with small through holes for the connector male seat, the soft flexible cable 23 and the image sensor chip 2 . The connection cables of the light source device 3 pass through and are connected to the external control device 5 , and silica gel is used for gas tightness at the small through hole. The connector female seat of the control device 5 is connected with the connector male seat, which is used to control the photosensitive area 21 of the image sensor chip 2 and the working mode of the light source device 3, and process the image or video data transmitted from the cell imaging device 6, and display it. As a result, as shown in Fig. 8, the real-time dynamic monitoring of the growth state of living cells can be realized in a full field of view without contacting and moving the sample. This system provides a large field of view while satisfying high resolution, and can monitor tens of thousands or even hundreds of thousands of cell samples at the same time, and can see the relative changes of different cell samples at the same time and at long distances. The continuous culture and real-time observation of live cell samples are realized, which greatly improves the efficiency of live cell culture observation. The system has a simple structure and simple operation, which greatly improves the portability and convenience of live cell culture and observation, and has broad application prospects.
实施例二 Embodiment 2
本发明还提供一种活细胞培养和实时观测方法，应用于上述任意一项的活细胞培养和实时观测系统，如图9所示，活细胞培养和实时观测方法包括以下步骤：The present invention also provides a live cell culture and real-time observation method, which is applied to any of the above-mentioned live cell culture and real-time observation systems. As shown in Figure 9, the live cell culture and real-time observation method includes the following steps:
步骤S1，将待培养和观测的活细胞和培养液接种至细胞培养皿内的图像传感器芯片的感光区上；Step S1, inoculating the live cells and culture solution to be cultured and observed on the photosensitive area of the image sensor chip in the cell culture dish;
步骤S2，对活细胞培养箱进行培养环境设置，并将接种有活细胞和培养液的图像传感器芯片和细胞培养皿，以及光源装置放置于活细胞培养箱内，并接入控制装置；Step S2, setting the culture environment for the living cell incubator, placing the image sensor chip and the cell culture dish inoculated with the living cells and the culture solution, and the light source device in the living cell incubator, and connecting to the control device;
步骤S3，控制装置控制光源装置开启，同时控制图像传感器芯片对活细胞样本的投影显微图像进行采集；Step S3, the control device controls the light source device to turn on, and simultaneously controls the image sensor chip to collect the projection microscopic image of the living cell sample;
步骤S4，控制装置在采集结束后接收采集得到的投影显微图像，并对投影显微图像进行处理后显示。In step S4 , the control device receives the projection microscopic image obtained by collection after the collection, and processes the projection microscopic image and displays it.
作为优选的实施方式，以091214原代胶质瘤细胞作为活细胞样本为例进行活细胞培养和实时观测，首先取5ml培养液90％DMED+10％FBS加入细胞培养皿中，并取091214原代胶质瘤细胞作 为活细胞样本接种至培养液内。随后通过活细胞培养箱中的温度控制器将活细胞培养箱中的环境温度设置为37℃，通过气体控制器将活细胞培养箱中的气体设置为5％CO2，95％O2，在活细胞培养箱中的活细胞培养环境稳定后，将图像传感器芯片和细胞培养皿，以及光源装置固定在活细胞培养箱内，将图像传感器芯片的连接器公座和软质排线，以及光源装置的连接排线穿过活细胞培养箱上预留的小型通孔，连接到外部的控制装置，关闭活细胞培养箱的箱门，同时保持培养箱的密封性。随后091214原代胶质瘤细胞在活细胞培养箱中进行持续不断地培养。As a preferred embodiment, taking 091214 primary glioma cells as a live cell sample for live cell culture and real-time observation, first add 5 ml of culture medium 90% DMED + 10% FBS to the cell culture dish, and take 091214 original The glioma cells were inoculated into the culture medium as live cell samples. Subsequently, the ambient temperature in the live cell incubator was set to 37°C by the temperature controller in the live cell incubator, and the gas in the live cell incubator was set to 5% CO2, 95% O2 by the gas controller, and in the live cell incubator After the living cell culture environment in the incubator is stabilized, fix the image sensor chip, cell culture dish, and light source device in the live cell incubator, connect the connector male seat and soft cable of the image sensor chip, and the light source device. Connect the cable through the small through hole reserved on the live cell incubator, connect it to the external control device, close the door of the live cell incubator, and keep the incubator airtight. The 091214 primary glioma cells were then continuously cultured in a live cell incubator.
作为优选的实施方式，控制装置上可以设有开始采集按钮，当需要对细胞培养皿内的活细胞样本进行观测时，点击开始采集按钮，控制装置给出采集信号，并控制光源装置开启，再通过控制图像传感器芯片开始采集投影显微图像，在采集结束后，控制装置控制光源装置关闭，并读取图像传感器芯片采集的投影显微图像，经处理后对投影显微图像进行显示，以供观测人员进行查看。As a preferred embodiment, the control device may be provided with a start acquisition button. When the live cell sample in the cell culture dish needs to be observed, click the start acquisition button, the control device will give a acquisition signal, and control the light source device to turn on, and then click the start acquisition button. By controlling the image sensor chip to start collecting the projection microscopic image, after the acquisition, the control device controls the light source device to turn off, and reads the projection microscopic image collected by the image sensor chip, and displays the projection microscopic image after processing for Observers to check.
作为优选的实施方式，执行步骤S1之前还包括：As a preferred embodiment, before performing step S1, it also includes:
对细胞培养皿、图像传感器芯片和光源装置进行灭菌处理，随后于细胞培养皿底部加促贴壁剂进行处理。Sterilize the cell culture dish, the image sensor chip and the light source device, and then add an adhesion promoting agent to the bottom of the cell culture dish for treatment.
作为优选的实施方式，灭菌处理的处理方式包括采用紫外光照射30分钟后，再采用无水酒精浸泡30分钟，随后静置10分钟直至表面完全干燥，随后在细胞培养皿底部加促贴壁剂处理30分钟后，将液体弃去，并用PBS清洗三次备用。上述促贴壁剂可以是左旋多聚赖 氨酸。As a preferred embodiment, the sterilization treatment method includes irradiating with ultraviolet light for 30 minutes, then soaking in anhydrous alcohol for 30 minutes, then standing for 10 minutes until the surface is completely dry, and then adding a wall to the bottom of the cell culture dish to promote adhesion After 30 minutes of treatment, the liquid was discarded and washed three times with PBS. The above-mentioned adhesion promoting agent may be L-polylysine.
作为优选的实施方式，步骤S3中，控制装置控制图像传感器芯片对活细胞样本的投影显微图像进行采集包括：As a preferred embodiment, in step S3, the control device controlling the image sensor chip to collect the projection microscopic image of the living cell sample includes:
控制装置控制图像传感器芯片以图像采集模式采集得到投影显微图像，以对活细胞样本的当前状态进行观测；或The control device controls the image sensor chip to acquire the projection microscopic image in the image acquisition mode, so as to observe the current state of the living cell sample; or
控制装置控制图像传感器芯片以视频采集模式采集得到预设视频采集时间的若干投影显微图像，以对活细胞样本在预设视频采集时间的细胞变化情况进行观测。The control device controls the image sensor chip to collect several projection microscopic images at the preset video collection time in the video collection mode, so as to observe the cell changes of the living cell sample at the preset video collection time.
以上所述仅为本发明较佳的实施例，并非因此限制本发明的实施方式及保护范围，对于本领域技术人员而言，应当能够意识到凡运用本说明书及图示内容所作出的等同替换和显而易见的变化所得到的方案，均应当包含在本发明的保护范围内。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the embodiments and protection scope of the present invention. Those skilled in the art should be aware of the equivalent replacements made by using the contents of the description and the drawings. The solutions obtained from obvious changes and obvious changes shall all be included in the protection scope of the present invention.

Claims (22)

  1. 一种活细胞培养和实时观测系统，其特征在于，具体包括：A live cell culture and real-time observation system, characterized in that it specifically includes:
    一细胞培养皿，用于承载活细胞样本和培养液；A cell culture dish for carrying live cell samples and culture medium;
    一图像传感器芯片，所述图像传感器芯片的正面与所述细胞培养皿的底面贴合，所述图像传感器芯片正面设有一感光区且所述细胞培养皿底部设有暴露所述感光区的开口，所述图像传感器芯片用于记录所述活细胞样本的投影显微图像；an image sensor chip, the front surface of the image sensor chip is attached to the bottom surface of the cell culture dish, a photosensitive area is formed on the front surface of the image sensor chip, and an opening exposing the photosensitive area is formed at the bottom of the cell culture dish, The image sensor chip is used to record the projection microscopic image of the living cell sample;
    一光源装置，固定于所述图像传感器芯片上方，所述光源装置用于为所述活细胞样本成像提供光源；a light source device fixed above the image sensor chip, the light source device is used to provide a light source for imaging the living cell sample;
    一活细胞培养箱，用于放置所述细胞培养皿、所述图像传感器芯片和所述光源装置，并为所述活细胞样本提供生长繁殖环境；a live cell incubator, used for placing the cell culture dish, the image sensor chip and the light source device, and providing a growth and reproduction environment for the live cell sample;
    一控制装置，分别连接所述图像传感器芯片和所述光源装置，用于控制所述图像传感器芯片和所述光源装置，并接收、处理和显示所述活细胞样本的所述投影显微图像。a control device, connected to the image sensor chip and the light source device respectively, for controlling the image sensor chip and the light source device, and receiving, processing and displaying the projection microscopic image of the living cell sample.
  2. 根据权利要求1所述的活细胞培养和实时观测系统，其特征在于，所述细胞培养皿包括设有所述开口的底板以及底部围合所述底板的侧壁，且所述底板的厚度≤500μm，所述侧壁的厚度≤500μm。The live cell culture and real-time observation system according to claim 1, wherein the cell culture dish comprises a bottom plate with the opening and a side wall surrounding the bottom plate at the bottom, and the thickness of the bottom plate is ≤ 500 μm, the thickness of the sidewall is ≤500 μm.
  3. 根据权利要求1所述的活细胞培养和实时观测系统，其特征在于，所述细胞培养皿的制作材料为透明玻璃，或透明的有机聚合物。The live cell culture and real-time observation system according to claim 1, wherein the cell culture dish is made of transparent glass or transparent organic polymer.
  4. 根据权利要求1所述的活细胞培养和实时观测系统，其特征在于，所述细胞培养皿的底面通过AB胶或紫外胶粘贴固定在所述图像传感器芯片的正面形成封闭浅槽，以承载所述活细胞样本和所述培养液。The live cell culture and real-time observation system according to claim 1, wherein the bottom surface of the cell culture dish is pasted and fixed on the front surface of the image sensor chip by AB glue or UV glue to form a closed shallow groove for carrying The living cell sample and the culture medium.
  5. 根据权利要求1所述的活细胞培养和实时观测系统，其特征在于，所述图像传感器芯片采用柔性印制电路板封装，且所述感光区周围的封装连接用金线采用芯片封装胶固定。The live cell culture and real-time observation system according to claim 1, wherein the image sensor chip is packaged with a flexible printed circuit board, and the package connection around the photosensitive area is fixed with a chip encapsulation glue.
  6. 根据权利要求5所述的活细胞培养和实时观测系统，其特征在于，所述柔性印制电路板通过一软质排线外接一连接器公座，且所述控制装置上设有与所述连接器公座适配的一连接器母座，所述图像传感器芯片通过所述软质排线、所述连接器公座和所述连接器母座与所述控制装置建立连接。The live cell culture and real-time observation system according to claim 5, wherein the flexible printed circuit board is connected to a male connector through a flexible cable, and the control device is provided with a A connector female seat adapted to the connector male seat, and the image sensor chip is connected with the control device through the flexible flat cable, the connector male seat and the connector female seat.
  7. 根据权利要求6所述的活细胞培养和实时观测系统，其特征在于，所述软质排线的基材为以聚酰亚胺或聚酯薄膜，双面加电子屏蔽膜，形状为长条型，厚度≤100μm。The living cell culture and real-time observation system according to claim 6, wherein the base material of the flexible flexible cable is a polyimide or polyester film, double-sided with an electronic shielding film, and the shape is a long strip Type, thickness ≤ 100μm.
  8. 根据权利要求6所述的活细胞培养和实时观测系统，其特征在于，所述连接器公座设置于一连接器底板的背面，所述连接器底板的基材为PCB软硬复合板且正面加钢片补强。The live cell culture and real-time observation system according to claim 6, wherein the connector male seat is arranged on the back of a connector base plate, and the base material of the connector base plate is a PCB soft and hard composite board and the front surface is Steel sheet reinforcement.
  9. 根据权利要求8所述的活细胞培养和实时观测系统，其特征在于，所述连接器公座的基材为金属，且所述连接器公座上设有一长矩形沟槽，所述长矩形沟槽两侧均匀分布有两排连接器引脚。The live cell culture and real-time observation system according to claim 8, wherein the base material of the connector male seat is metal, and the connector male seat is provided with a long rectangular groove, and the long rectangular groove is formed on the connector male seat. There are two rows of connector pins evenly distributed on both sides of the groove.
  10. 根据权利要求1所述的活细胞培养和实时观测系统，其特征在于，所述图像传感器芯片采用半浮栅晶体管或复合介质栅光敏探测器作为感光像素单元。The live cell culture and real-time observation system according to claim 1, wherein the image sensor chip adopts a semi-floating gate transistor or a composite dielectric gate photosensitive detector as a photosensitive pixel unit.
  11. 根据权利要求10所述的活细胞培养和实时观测系统，其特征在于，所述图像传感器芯片的单个所述感光像素单元的尺寸≤ 500mm×500nm，所述图像传感器芯片的所述感光像素单元的数目≥4亿。The live cell culture and real-time observation system according to claim 10, wherein the size of a single photosensitive pixel unit of the image sensor chip is ≤ 500mm×500nm, and the size of the photosensitive pixel unit of the image sensor chip Number ≥ 400 million.
  12. 根据权利要求1所述的活细胞培养和实时观测系统，其特征在于，所述光源装置包括：The live cell culture and real-time observation system according to claim 1, wherein the light source device comprises:
    光源主板，所述光源主板上集成一红绿蓝三色LED光源；a light source mainboard, which integrates a red, green and blue three-color LED light source;
    若干可调节支架，分别固定于所述光源主板的朝向所述红绿蓝三色LED光源的一侧，各所述可调节支架用于支撑和固定所述光源主板并调节所述光源主板的照明高度。Several adjustable brackets are respectively fixed on the side of the light source main board facing the red, green and blue LED light sources, and each adjustable bracket is used to support and fix the light source main board and adjust the illumination of the light source main board high.
  13. 根据权利要求12所述的活细胞培养和实时观测系统，其特征在于，所述红绿蓝三色LED光源通过一连接排线与所述控制装置连接，以通过所述控制装置控制所述红绿蓝三色LED光源的工作模式和照明亮度。The live cell culture and real-time observation system according to claim 12, wherein the red, green and blue LED light source is connected to the control device through a connecting cable, so as to control the red, green and blue light through the control device. Working mode and lighting brightness of green and blue LED light source.
  14. 根据权利要求1所述的活细胞培养和实时观测系统，其特征在于，所述活细胞培养箱的外部设有保温层。The live cell culture and real-time observation system according to claim 1, wherein a thermal insulation layer is provided on the outside of the live cell incubator.
  15. 根据权利要求1所述的活细胞培养和实时观测系统，其特征在于，所述活细胞培养箱的内部设有一托盘，用于放置所述细胞培养皿、所述图像传感器芯片和所述光源装置。The live cell culture and real-time observation system according to claim 1, wherein a tray is provided inside the live cell incubator for placing the cell culture dish, the image sensor chip and the light source device .
  16. 根据权利要求1所述的活细胞培养和实时观测系统，其特征在于，所述活细胞培养箱内部设有温度控制器和气体控制器，通过所述温度控制器和所述气体控制器调节所述活细胞培养箱中的温度和气体浓度，为所述活细胞样本提供生长繁殖环境。The live cell culture and real-time observation system according to claim 1, wherein a temperature controller and a gas controller are provided inside the live cell incubator, and the temperature controller and the gas controller are used to adjust all the The temperature and gas concentration in the live cell incubator provide a growth and reproduction environment for the live cell sample.
  17. 根据权利要求1所述的活细胞培养和实时观测系统，其特征 在于，所述控制装置设置于所述活细胞培养箱的外部，所述活细胞培养箱的箱体背面设有小型通孔，用于供所述控制装置分别与所述图像传感器芯片和所述光源装置的连接走线。The live cell culture and real-time observation system according to claim 1, wherein the control device is arranged outside the live cell incubator, and the back of the box body of the live cell incubator is provided with a small through hole, The wiring is used for connecting the control device with the image sensor chip and the light source device respectively.
  18. 一种活细胞培养和实时观测方法，其特征在于，应用于如权利要求1-17中任意一项所述的活细胞培养和实时观测系统，所述活细胞培养和实时观测方法包括以下步骤：A kind of living cell culture and real-time observation method, is characterized in that, is applied to the living cell culture and real-time observation system as described in any one in claim 1-17, and described living cell culture and real-time observation method comprise the following steps:
    步骤S1，将待培养和观测的活细胞和培养液接种至所述细胞培养皿内的所述图像传感器芯片的所述感光区上；Step S1, inoculating live cells and culture liquid to be cultured and observed on the photosensitive area of the image sensor chip in the cell culture dish;
    步骤S2，对所述活细胞培养箱进行培养环境设置，并将接种有所述活细胞和所述培养液的所述图像传感器芯片和所述细胞培养皿，以及所述光源装置放置于所述活细胞培养箱内，并接入所述控制装置；Step S2, setting the culture environment for the living cell incubator, and placing the image sensor chip and the cell culture dish inoculated with the living cells and the culture medium, and the light source device on the in the living cell incubator, and connected to the control device;
    步骤S3，所述控制装置控制所述光源装置开启，同时控制所述图像传感器芯片对所述活细胞样本的所述投影显微图像进行采集；Step S3, the control device controls the light source device to turn on, and simultaneously controls the image sensor chip to collect the projection microscopic image of the living cell sample;
    步骤S4，所述控制装置在采集结束后接收采集得到的所述投影显微图像，并对所述投影显微图像进行处理后显示。In step S4, the control device receives the projection microscopic image obtained after collection, and processes the projection microscopic image and displays it.
  19. 根据权利要求18所述的活细胞培养和实时观测方法，其特征在于，执行所述步骤S1之前还包括：The live cell culture and real-time observation method according to claim 18, characterized in that, before performing the step S1, it further comprises:
    对所述细胞培养皿、所述图像传感器芯片和所述光源装置进行灭菌处理，随后于所述细胞培养皿底部加促贴壁剂进行处理。Sterilize the cell culture dish, the image sensor chip and the light source device, and then add an adhesion promoting agent to the bottom of the cell culture dish for treatment.
  20. 根据权利要求19所述的活细胞培养和实时观测方法，其特征在于，所述灭菌处理的处理方式包括采用紫外光照射和采用无水酒 精浸泡。live cell culture and real-time observation method according to claim 19, is characterized in that, the treatment mode of described sterilization treatment comprises adopting ultraviolet light irradiation and adopting absolute alcohol immersion.
  21. 根据权利要求18所述的活细胞培养和实时观测方法，其特征在于，所述步骤S3中，所述控制装置控制所述图像传感器芯片对所述活细胞样本的所述投影显微图像进行采集包括：The method for culturing and real-time observation of living cells according to claim 18, wherein in step S3, the control device controls the image sensor chip to collect the projection microscopic image of the living cell sample include:
    所述控制装置控制所述图像传感器芯片以图像采集模式采集得到所述投影显微图像，以对所述活细胞样本的当前状态进行观测；或The control device controls the image sensor chip to acquire the projection microscopic image in an image acquisition mode, so as to observe the current state of the living cell sample; or
    所述控制装置控制所述图像传感器芯片以视频采集模式采集得到预设视频采集时间的若干所述投影显微图像，以对所述活细胞样本在所述预设视频采集时间的细胞变化情况进行观测。The control device controls the image sensor chip to collect a plurality of the projection microscopic images at a preset video collection time in a video collection mode, so as to perform cell changes of the living cell sample at the preset video collection time. observation.
  22. 根据权利要求18所述的活细胞培养和实时观测方法，其特征在于，所述活细胞培养箱内设有温度控制器和气体控制器，所述步骤S2中，通过所述温度控制器和所述气体控制器对所述活细胞培养箱进行培养环境设置。The method for culturing living cells and real-time observation according to claim 18, wherein a temperature controller and a gas controller are provided in the living cell incubator, and in the step S2, the temperature controller and the The gas controller sets the culture environment for the living cell incubator.
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