CN112210497A - Long-time living cell incubation system combined with LED array and optogenetic living cell imaging method - Google Patents
Long-time living cell incubation system combined with LED array and optogenetic living cell imaging method Download PDFInfo
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Abstract
The invention discloses a living cell long-time incubation system combined with an LED array and a optogenetics living cell imaging method. The system is used in a living cell workstation, and provides a portable and stable culture environment for long-time living cell imaging experiments. Besides a gas supply system, a temperature control system and a closed culture cabin which provide stable culture conditions for cells, the system is additionally provided with an LED array for assisting optogenetics experiments. The LED array is formed by alternately arranging LED lamp beads with the emission wavelengths of 488nm (blue light), 561nm (green light) and 640nm (red light), and the light-emitting wavelength of the LED array can be changed in real time as required. The light emitting mode of the LED array is changed by the controller, and the current working state of the LED array is displayed in real time. The light emitted by the LED array finally horizontally irradiates the culture dish in the center of the closed culture cabin, and the imaging light path of the living cell workstation cannot be influenced.
Description
Technical Field
The invention relates to the technical fields of optogenetics, circuit electronics, microimaging, optics and the like, in particular to a living cell long-time incubation system combined with an LED array and an optogenetics living cell imaging method.
Technical Field
Optogenetics is a new subject, integrates related knowledge such as optics and biology and related technologies such as genetic engineering and software engineering, can control various life activities of organisms in a light regulation mode, and has a series of advantages of no damage, non-invasion, simple and convenient operation, accurate regulation, high resolution, high repeatability and the like.
Microscopic imaging techniques have long been used in the field of bioscience, and researchers have labeled structures (such as mitochondria, vesicles, etc.) participating in life activities in cells with fluorescent proteins or dyes, and then have studied the life processes of cells by photographing and observing the positions of the labeled structures in the cells with the microscopic imaging techniques. By continuously shooting a plurality of microscopic pictures of living cells, synthesizing a video and tracking the motion track of a marked structure, the method can more clearly understand the proceeding mode of the life activities of the cells. However, many of the life processes of a cell often take several hours or even days to be fully developed. In order to continuously observe the life activities of living cells for a long period of time, it is necessary to provide a stable culture environment for the cells.
The living cell workstation can realize that the living cells are subjected to microscopic imaging under the condition of simulating in-vivo environment in vitro, and the living cell long-time incubation system keeps the stability of the cell culture state. One of the major challenges faced in live cell imaging is how to maintain the viability of the cells during the course of the experiment and to make the cells function as close to natural as possible.
In addition, the long-time living cell incubation system in the traditional living cell workstation lacks a stimulating light source, so that the research of optogenetic cell imaging cannot be carried out, and an external light source often interferes with an imaging light path to influence the imaging effect. The optogenetic technology is an important technology that can be used to control the vital movements of cells, and in order to more accurately study the details of the vital movements of cells, a long-time living cell incubation system capable of realizing optogenetic cell study is needed, and an imaging optical path that does not affect a living cell workstation is also needed, which is a great challenge in the optogenetic imaging field.
Disclosure of Invention
In order to solve the problems and make up the defects of a long-time optogenetic imaging experimental device, the invention provides a living cell long-time incubation system combined with an LED array and an optogenetic living cell imaging method. The living cell long-time incubation system provided by the invention is composed of a gas supply system, a temperature control system, a closed culture cabin, an LED array, a mirror projection system and an LED array controller. The LED array is formed by alternately arranging LED lamp beads with the emission wavelengths of 488nm (blue light), 561nm (green light) and 640nm (red light), and the light-emitting wavelength of the LED array can be changed in real time as required. The light emitting mode of the LED array is changed by the controller, and the current working state of the LED array is displayed on the controller in real time. The light emitted by the LED array horizontally irradiates a culture dish in the center of the closed culture cabin after being reflected by the concave mirror, and the imaging light path of the living cell workstation cannot be influenced. The system is completely suitable for a living cell workstation, and provides a portable and stable culture environment for long-time optogenetic living cell imaging.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a living cell long-time incubation system combined with an LED array comprises a gas supply system, a temperature control system, a closed culture cabin, the LED array, a mirror projection system and an illumination controller;
a transparent window for light transmission imaging is arranged on the outer wall of the closed culture cabin, and an object stage is arranged at the center of the inside of the closed culture cabin; the gas supply system and the temperature control system respectively control the gas environment and the temperature in the closed culture cabin;
the LED array is formed by circularly and alternately arranging a plurality of LED lamp beads with different wavelengths, uniformly surrounds the inner wall of the closed culture cabin in a circumferential mode and is positioned at the periphery of the objective table;
the mirror projection system surrounds one side, far away from the objective table, of the LED array, and light emitted from the LED array is reflected by the mirror projection system and then projected to the objective table in the closed culture cabin respectively in parallel light in the horizontal direction;
the illumination controller comprises a display panel, an adjusting panel and a wireless communication module, and the wireless communication module is wirelessly connected with the LED array; LED parameters are set through the adjusting panel, the parameters are transmitted to the LED array through the wireless communication module, and the working state of the LED array is displayed in real time through the display panel.
Preferably, the temperature control system consists of a temperature sensor, a temperature controller and a water bath system, wherein the temperature sensor measures the temperature in the closed cabin in real time, and the water bath system is started to heat when the measured temperature is lower than the lower limit of the preset temperature; and when the measured temperature is higher than the preset upper temperature limit, starting the water bath system to dissipate heat.
Preferably, the water bath system is installed on the outer wall of the closed culture chamber, and is cooled by a circulating water cooling method and heated by a circulating water heating method.
Preferably, the LED array is formed by circularly and alternately arranging three LED lamp beads with different wavelengths, namely blue LED lamp beads with the wavelength of 488nm, green LED lamp beads with the wavelength of 561nm and red LED lamp beads with the wavelength of 640 nm.
Preferably, the LED lamp beads with each wavelength in the LED array can be controlled independently.
As the optimization of the invention, the closed culture cabin is designed to be double-layer, wherein a water bath layer is arranged between the inner layer and the outer layer, and a water inlet and a water outlet are arranged on the water bath layer.
Preferably, the mirror projection system is composed of a series of concave mirrors, the number of the concave mirrors is the same as that of the LED lamp beads in the LED array, and each LED lamp bead is located at the focal point of the corresponding concave mirror.
Preferably, each LED lamp bead is provided with a shading layer facing to the semi-circle surface of the center of the closed culture cabin.
Preferably, the LED parameters include lighting state, lighting time, illumination intensity and illumination frequency.
Another object of the present invention is to provide a method for optogenetically imaging living cells using the above system, comprising the steps of:
1) enabling living cells to express specific photosensitive protein through transfection, and inoculating the transfected living cells into a perforated culture dish for imaging; marking the intracellular structure of the living cell to be observed by a living cell staining method after the cell adheres to the wall and expresses the corresponding photosensitive protein;
2) starting the living cell long-time incubation system, and setting gas environment parameters, temperature parameters and LED parameters suitable for living of living cells through a regulating panel; the wireless communication module transmits the set LED parameters to the LED array, controls the LED lamp beads needing to be lightened, the lightening time, the irradiation intensity and the irradiation frequency, and feeds back the working state of each LED lamp bead to the display panel in real time for visual display;
the gas supply system is used for introducing gas with preset concentration into the closed culture cabin according to the gas environment parameters; the temperature control system controls the temperature in the closed culture cabin to be in a preset interval according to the temperature parameters;
3) placing the perforated culture dish loaded with living cells to be observed on an object stage in a closed culture cabin, wherein a semi-circular surface far away from the center of the closed culture cabin in each lighted LED lamp bead emits divergent light, and each light is reflected by a corresponding concave surface in a mirror projection system to form parallel light to irradiate the cells to be observed on the object stage;
4) and placing the closed culture cabin on a microscope objective table, aligning a transparent window on the outer wall of the closed culture cabin with the objective lens, setting imaging parameters of the microscope, and recording the life movement track of the living cells to be observed.
Compared with the prior art, the invention has the advantages that:
1) in order to enable the traditional living cell workstation to meet the long-time imaging function of optogenetics living cells, the invention provides a living cell long-time incubation system combined with an LED array, which comprises a gas supply system for providing stable culture conditions for cells, a temperature control system, a closed culture cabin and the LED array for assisting optogenetics experiments. The light emitting mode of the LED array can be changed by the controller, the system is stable, and the requirement of long-time imaging experiments is met;
2) the LED array is used as a light source, is formed by circularly and alternately arranging a plurality of LED lamp beads with different wavelengths, uniformly surrounds the inner wall of the closed culture cabin in a circumferential mode, and can change the light-emitting wavelength of the LED array in real time according to the requirement; because the LED lamp beads are point light sources, the light emitted by the LED lamp beads directly irradiates a sample and can influence the light path of an imaging system, and an operation control system for turning off the light source during imaging is complex and can influence the life activity of living cells.
3) The method adopts the optogenetic technology to control the life activities of the cells, utilizes the living cell workstation of the long-time living cell incubation system combined with the LED array to track the life activity process of the cells, combines the living cell workstation and the LED array to more accurately study all details of the life activities, meets the requirements of different experimental conditions, realizes the full-automatic acquisition of the life activity track of the optogenetic living cells, and is simple and convenient to operate.
Drawings
FIG. 1 is a schematic cross-sectional view of a living cell long-term incubation system in the present embodiment;
FIG. 2 is a schematic view showing the positional relationship of the LED array, the mirror projection system, and the stage in the present embodiment;
reference numerals: a gas supply system 1; a temperature sensor 201, a temperature controller 202 and a water bath system 203; a closed culture chamber 3, a transparent window 301 and an object stage 302; the LED lamp comprises an LED array 4, LED lamp beads 401 and a light shielding layer 402; a mirror projection system 5, a concave mirror 501; illumination controller 6, wireless communication module 601.
Detailed Description
The invention will be further described with reference to the accompanying drawings.
The invention provides a living cell long-time incubation system combined with an LED array, as shown in figure 1, comprising: a gas supply system 1, a temperature control system, a closed culture chamber 3, an LED array 4, a mirror projection system 5, and an illumination controller 6. A transparent window 301 for light-transmitting imaging is arranged on the outer wall of the closed culture cabin 1; the gas supply system 1 and the temperature control system respectively control the gas environment and the temperature in the closed culture chamber 3;
the LED array 4 is formed by circularly and alternately arranging LED lamp beads 401 with various wavelengths, uniformly surrounds the inner wall of the closed culture cabin in a circumferential mode, and is positioned on the periphery of the objective table 302;
the mirror projection system 5 surrounds one side, far away from the objective table, of the LED array 4, and light emitted from the LED array is projected onto the objective table in the closed culture cabin respectively in parallel light in the horizontal direction after being reflected by the mirror projection system;
the illumination controller 6 comprises a display panel, an adjusting panel and a wireless communication module 601, wherein the wireless communication module 601 is wirelessly connected with the LED array 4; the LED parameters are set through the adjusting panel, the parameters are transmitted to the LED array 4 through the wireless communication module 601, and the working state of the LED array is displayed in real time through the display panel.
In one embodiment of the invention, the gas supply system provides carbon dioxide to the enclosure to maintain the concentration of carbon dioxide in the enclosure at a concentration (typically 5% and adjustable) that is optimal for cell growth. The gas concentration adjusting process comprises the following steps: the gas concentration in the cabin measured by the sensor is compared with the set concentration, and the gas concentration in the cabin is kept near the set value by inputting the specific gas, so that the cells are ensured to be in the optimal growth state.
The temperature control system consists of a temperature sensor 201, a temperature controller 202 and a water bath system 203. Due to the existence of the LED array and the LED control system for optogenetic experiments in the incubation system, although the LED belongs to a cold light source, the loss of the circuit during operation can be inevitably converted into heat. This results in that even if the temperature in the chamber is not heated for a long time, the temperature in the chamber may exceed a set value to cause the culture environment of the cells to deviate from the optimum state, and the temperature in the chamber may become too high to affect the activity of the cells. It is therefore necessary to take temperature reduction measures in the temperature control system. Specifically, the temperature sensor measures the temperature in the closed cabin in real time, and when the measured temperature is lower than a preset lower temperature limit (usually 37 ℃), the water bath system is started to heat; and when the measured temperature is higher than the preset upper temperature limit, starting the water bath system to dissipate heat.
In one specific implementation of the invention, the water cooling system is adopted to cool the temperature, when the temperature sensor detects that the temperature in the cabin is higher than a set value and exceeds a threshold value, the water cooling system starts to work, the cooling water at room temperature is conveyed into the water bath layer on the outer layer of the cabin, and after the temperature of the water in the water bath layer rises, new cooling water at room temperature is conveyed into the water bath layer, so that the purpose of lowering the temperature in the cabin is realized in a circulating manner. When the temperature in the cabin is too low, the cooling water passing through the heating water bath layer becomes heating water, and the temperature in the cabin is increased in a water bath mode.
The closed chamber of the present invention provides a closed steady-state environment for cell culture, and the gas concentration and temperature inside the chamber are stabilized in the optimum cell growth state (which can be set according to specific experimental requirements) by the gas supply system 1 and the temperature control system. The cabin is respectively provided with a water bath layer, a mirror projection system and an LED array from outside to inside. The water bath layer is positioned at the outermost side of the whole cabin and is used for keeping the temperature in the cabin stable at the temperature most suitable for cell growth. The LED array is used as a light source for long-time optogenetic experiments, and the mirror projection system irradiates light emitted by the LED array to the center of the cabin in parallel light. In the experiment, a sample to be observed is placed in the center of a cabin, and long-time live cell imaging is carried out under an environment suitable for cell culture. The whole cabin is small in size and can be directly placed on a microscope objective table of a living cell workstation, and the central parts of the bottom and the top are made of transparent materials so as to facilitate imaging. The top lid can be opened by sliding to facilitate handling of the sample.
The LED array is positioned on the inner side of the cabin and is arranged in a row in a circumferential mode to surround the side wall of the cabin. The LED array is used as a light source of optogenetic experiments, in order to use one set of device to deal with the experimental conditions as many as possible, the LED array is composed of three LED lamp beads with different wavelengths, namely 488nm (blue), 561nm (green) and 640nm (red), and the requirements of various light control elements on the wavelength of excitation light are met as much as possible. LED lamp beads with three wavelengths are circularly arranged on a cabin wall in a mode of '… -blue-green-red- …', and LED lamp beads with any color are arranged in a row in a circular mode and evenly surround the side wall of the cabin.
The mirror projection system 5 is located on the inner wall of the cabin, at the periphery of the LED array. The function of the LED is to irradiate a point light source emitted by the LED to a sample positioned in the center of the cabin in a horizontal direction in parallel by the reflection principle of the concave mirror.
The mirror projection system is used as an auxiliary system of the LED array and is used for horizontally irradiating the light emitted by the LED lamp beads on a sample positioned in the center of the cabin after the light path is converted. As shown in fig. 2, the LED lamp beads can be regarded as point light sources, the light emitted by the LED lamp beads directly irradiates a sample and may affect the light path of the imaging system, and the operation control system for turning off the light source during imaging is complex and may affect the life activity of living cells, so that the originally divergent point light sources are converted into parallel light to irradiate the center of the cabin through the reflection principle of the concave mirror. All LED lamp beads 401 on the LED array face the semi-circle surface of the center of the cabin to be provided with a light shielding layer 402, for example, opaque black paint can be sprayed, and the influence of light on the side on imaging is eliminated. A concave mirror 501 is arranged at the corresponding position behind each LED lamp bead, and the LED lamp beads are just positioned at the focus of the concave mirror, so that the light on the side is reflected by the concave mirror and then becomes parallel light in the horizontal direction to irradiate a sample positioned at the center of the cabin.
The illumination controller 6 comprises a display panel, an adjusting panel and a wireless communication module 601, wherein the wireless communication module 601 is wirelessly connected with the LED array 4; the LED parameters are set through the adjusting panel, the parameters are transmitted to the LED array 4 through the wireless communication module 601, and the working state of the LED array is displayed in real time through the display panel.
Specifically, the display panel displays current LED illumination parameters (whether LEDs of each color are turned on or not, the turn-on time, the illumination intensity, the illumination frequency and the like) in real time, the adjusting panel provides adjusting options for operators to use, and the illumination mode of lamp beads of each color on the LED array is adjusted so as to meet the requirements on light sources under different optogenetics experiment conditions. The wireless communication module 601 transmits the determined illumination parameters on the adjusting panel to the LED array to control the LED array to work.
A typical experimental procedure for optogenetic live cell imaging using the present system is as follows:
cells are made to express specific light sensitive proteins by transfection, and the transfected cells are seeded in imaging perforated dishes. After the cells are adherent and express the corresponding light sensitive protein, the intracellular structures to be observed are marked by methods such as staining of viable cells (the cells may also be allowed to express the corresponding fluorescent protein at the transfection step). After setting the operating parameters of the long-term living cell incubation system (typically 5% carbon dioxide, 37 ℃) the perforated culture dish is placed in the chamber and placed on the stage of the microscope. And finally, after the imaging parameters of the microscope are set, the living cell workstation can automatically record the life activity track of the sample in the experimental process for an experimenter to study.
In the operation process, the gas supply system, the temperature control system and the closed culture cabin provide stable culture conditions for living cells, the LED array provides a light source for the implementation of the optogenetic technology, and gas environment parameters, temperature parameters and LED parameters suitable for living of the living cells are set through the adjusting panel;
the gas supply system is used for introducing gas with preset concentration into the closed culture cabin according to the gas environment parameters;
the temperature control system controls the temperature in the closed culture cabin to be in a preset interval according to the temperature parameters;
the wireless communication module 601 adjusts the LED lamp beads to be lightened, the lightening time, the irradiation intensity and the irradiation frequency through the LED parameters, and feeds back the working state of the LED array to the display panel in real time for visual display.
In the process of recording the life activity track of the living cells in real time, divergent light rays emitted from the semi-circular surface far away from the center of the closed culture cabin in each lighted LED lamp bead are reflected by the mirror projection system to form parallel illumination to the cells to be observed, so that the normal operation of the life activity of the living cells is ensured.
In conclusion, the invention adopts the optogenetic technology to control the life activities of the cells, and utilizes the living cell workstation of the long-time living cell incubation system combined with the LED array to track the life activity process of the cells, and combines the living cell workstation and the living cell workstation to more accurately study the details of the life activities.
The foregoing lists merely illustrate specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (10)
1. A long-time living cell incubation system combined with an LED array is characterized by comprising a gas supply system (1), a temperature control system, a closed culture cabin (3), the LED array (4), a mirror projection system (5) and an illumination controller (6);
a transparent window (301) for light-transmitting imaging is arranged on the outer wall of the closed culture cabin (3), and an object stage (302) is arranged at the center position in the closed culture cabin (3); the gas supply system (1) and the temperature control system respectively control the gas environment and the temperature in the closed culture chamber (3);
the LED array (4) is formed by circularly and alternately arranging a plurality of LED lamp beads (401) with different wavelengths, uniformly surrounds the inner wall of the closed culture cabin in a circumferential mode, and is positioned on the periphery of the objective table (302);
the mirror projection system (5) surrounds one side, far away from the objective table, of the LED array (4), and light emitted from the LED array is projected onto the objective table in the closed culture cabin in a horizontal direction after being reflected by the mirror projection system;
the illumination controller (6) comprises a display panel, an adjusting panel and a wireless communication module (601), wherein the wireless communication module (601) is in wireless connection with the LED array (4); LED parameters are set through the adjusting panel, the parameters are transmitted to the LED array (4) through the wireless communication module (601), and the working state of the LED array is displayed in real time through the display panel.
2. The long-time living cell incubation system combined with the LED array as claimed in claim 1, wherein the temperature control system is composed of a temperature sensor (201), a temperature controller (202) and a water bath system (203), the temperature sensor measures the temperature in the closed chamber in real time, and when the measured temperature is lower than a preset lower temperature limit, the water bath system is started to heat; and when the measured temperature is higher than the preset upper temperature limit, starting the water bath system to dissipate heat.
3. The long-time living cell incubation system combined with the LED array as claimed in claim 2, wherein the water bath system is installed on the outer wall of the closed culture chamber, and is cooled by a circulating water cooling method and heated by a circulating water heating method.
4. The system for long-time incubation of living cells combined with the LED array as claimed in claim 1, wherein the LED array is composed of three LED lamp beads (401) with different wavelengths which are circularly and alternately arranged, namely blue LED lamp beads with the wavelength of 488nm, green LED lamp beads with the wavelength of 561nm and red LED lamp beads with the wavelength of 640 nm.
5. The long-term living cell incubation system with LED array as claimed in claim 1, wherein LED beads of each wavelength in the LED array can be controlled individually.
6. The long-term living cell incubation system combined with the LED array as claimed in claim 1, wherein the closed culture chamber is a double-layer design, wherein a water bath layer is arranged between the inner layer and the outer layer, and the water bath layer is provided with a water inlet and a water outlet.
7. A long-term living cell incubation system combined with an LED array according to claim 1, wherein the mirror projection system is composed of a series of concave mirrors (501), the number of concave mirrors is the same as the number of LED beads (401) in the LED array, and each LED bead is located at the focal point of the corresponding concave mirror.
8. The long-time living cell incubation system combined with the LED array as claimed in claim 1, wherein the semi-circular surface of each LED lamp bead facing the center of the closed culture chamber is provided with a light shielding layer (402).
9. The long-term living cell incubation system according to claim 1, wherein the LED parameters include illumination status, illumination duration, illumination intensity and illumination frequency.
10. A method for optogenetically live cell imaging using the system of any one of claims 1-9, comprising the steps of:
1) enabling living cells to express specific photosensitive protein through transfection, and inoculating the transfected living cells into a perforated culture dish for imaging; marking the intracellular structure of the living cell to be observed by a living cell staining method after the cell adheres to the wall and expresses the corresponding photosensitive protein;
2) starting the living cell long-time incubation system of any one of claims 1-9, setting the gas environment parameters, the temperature parameters and the LED parameters suitable for living of the living cells by adjusting the panel; the wireless communication module (601) transmits the set LED parameters to the LED array, controls the LED lamp beads needing to be lightened, the lightening time, the illumination intensity and the illumination frequency, and feeds back the working state of each LED lamp bead to the display panel in real time for visual display;
the gas supply system (1) introduces gas with preset concentration into the closed culture cabin according to the gas environment parameters;
the temperature control system controls the temperature in the closed culture cabin to be in a preset interval according to the temperature parameters;
3) placing a perforated culture dish loaded with living cells to be observed on an object stage (302) in a closed culture cabin, emitting divergent light rays from a semicircular surface, far away from the center of the closed culture cabin (3), of each lighted LED lamp bead (401), and reflecting each light ray by a corresponding concave mirror (501) in a mirror projection system (5) to form parallel light to the cells to be observed on the object stage;
4) the closed culture cabin is placed on a microscope objective table, a transparent window (301) on the outer wall of the closed culture cabin is aligned to an objective lens, imaging parameters of a microscope are set, and the life activity track of living cells to be observed is recorded.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117070349A (en) * | 2023-08-28 | 2023-11-17 | 杭州诚佑生物科技有限公司 | Culture dish for improving activity of stem cells based on red light irradiation principle |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117070349A (en) * | 2023-08-28 | 2023-11-17 | 杭州诚佑生物科技有限公司 | Culture dish for improving activity of stem cells based on red light irradiation principle |
| CN117070349B (en) * | 2023-08-28 | 2024-01-19 | 杭州诚佑生物科技有限公司 | Culture dish for improving activity of stem cells based on red light irradiation principle |
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