CN111122525B - Fluorescence-patch clamp-micro suction tube detection device - Google Patents
Fluorescence-patch clamp-micro suction tube detection device Download PDFInfo
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- CN111122525B CN111122525B CN201911267029.5A CN201911267029A CN111122525B CN 111122525 B CN111122525 B CN 111122525B CN 201911267029 A CN201911267029 A CN 201911267029A CN 111122525 B CN111122525 B CN 111122525B
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
Abstract
The invention discloses a fluorescence-patch clamp-micropipette detection device. An experiment cavity, a three-dimensional micromanipulator and a patch clamp are arranged on the experiment platform, and the two sides of the experiment cavity are hollowed out for a micro suction pipe and a glass electrode to enter; the experiment cavity is provided with fluorescent cells, the micro-suction tube sucks red blood cells, the cells and the red blood cells are in extracellular fluid in the experiment cavity, and electrode fluid is filled in the glass electrode; the patch clamp comprises a patch clamp amplifier, a patch clamp probe and a patch clamp probe bracket; the full spectrum light emitted by the fluorescence light source forms fluorescence incident light through the color filter, the fluorescence incident light enters cells in the experiment cavity, and the fluorescence of the cells returns to be received by the fluorescence camera. The invention can collect the fluorescence image with high signal-to-noise ratio to research the transmembrane signal conduction of the membrane protein while detecting the influence of the potential change of the cell membrane on the interaction of the membrane protein, and can record the coupling relation among three spectral phase information while synchronously recording three spectral phase information such as a fluorescence spectrum, an electrophysiological spectrum, an adhesion state spectrum and the like.
Description
Technical Field
The invention relates to the integration of a fluorescence imaging technology, a patch clamp technology and a micropipette technology, in particular to a detection device capable of detecting the regulation rule of interaction between membrane potential and protein molecules and researching transmembrane signal conduction of membrane protein.
Background
The membrane potential of the cell membrane is a key regulatory factor for the vital activities of the cell. The membrane potential of the neuron plays an important role in regulating and controlling various vital activities of the neuron, and is an important biophysical factor for dynamically regulating and controlling the structure and function of a brain neural network; in non-neuronal cells, the membrane potential of the cell also regulates its vital activities such as proliferation and differentiation. The patch clamp technology is an effective means for researching the problems related to the membrane potential, and the whole-cell recording mode of the patch clamp technology can realize accurate and rapid control of the membrane potential of the whole cell membrane. However, patch clamping is currently primarily limited to studies of ion channel characteristics.
The membrane protein is the main receptor for the cell to sense the external environment and respond, and how most protein molecules on the cell membrane sense the membrane potential change to adjust the dynamic function of the cell membrane is not analyzed. The major bottleneck is the lack of direct and effective research means.
Micropipette technology is widely used for in situ detection of kinetic parameters of protein-protein interaction. In the detection process of the micropipette experiment, protein molecules are constantly in the microenvironment of the cell membrane (the protein molecules to be detected are respectively connected and expressed on erythrocytes and cell surfaces). Therefore, the micropipette experiment has the advantage of being unique in detecting the interaction between the membrane receptor and the ligand. The fusion of the patch clamp technology and the micropipette technology provides possibility for researching the dynamic regulation and control of the membrane potential change on the interaction between the membrane proteins under physiological conditions.
In addition, the fluorescence imaging technology is widely applied to the research of transmembrane signal transduction of membrane proteins, and only the integration of the patch clamp technology into the micropipette technology can only research the influence of membrane potential change on the interaction of the membrane proteins, but cannot research the influence of the membrane potential change on the transmembrane signal of the membrane proteins. Therefore, the transmembrane signal transduction of the membrane protein can be further researched by further integrating the fluorescence imaging technology on the basis of the integration of the patch clamp technology. The fluorescence imaging-patch clamp-micropipette technology which integrates three spectral phase information of fluorescence spectrum, electrophysiological spectrum and adhesion frequency can detect the influence of the potential change of the cell membrane on the interaction of the membrane protein and simultaneously observe the influence of the potential change of the cell membrane on the transmembrane signal conduction of the membrane protein in real time.
Disclosure of Invention
In order to solve the technical problems mentioned in the background art, the invention aims to provide a fluorescence-patch clamp-micropipette detection device which can detect the influence of the potential change of a cell membrane on the interaction of membrane proteins and simultaneously observe the influence of the potential change of the cell membrane on the transmembrane signal conduction of the membrane proteins in real time. The invention can synchronously record three spectral phase information such as fluorescence spectrum, electrophysiological spectrum, adhesion state spectrum and the like and simultaneously record the coupling relation among the three spectral phase information.
In order to achieve the purpose, the invention adopts the technical scheme that:
the system comprises an experiment platform, a bright-field mercury lamp light source, an inverted microscope, a glass electrode, a micro-suction pipe, a piezoelectric motion module, a first three-dimensional micromanipulator, a recording electrode and an experiment cavity framework.
An experiment cavity, a first three-dimensional micromanipulator, a patch clamp probe bracket and the like are arranged on the experiment platform, an experiment cavity framework is positioned in the center of the experiment platform, an upper glass sheet and a lower glass sheet are placed on the experiment cavity framework, an experiment cavity is formed between the two glass sheets, and the two sides of the experiment cavity are hollowed out for a micro-suction tube and a glass electrode to enter; the bottom surface of the experiment cavity is provided with a fluorescent cell in a whole cell recording mode, the micropipette absorbs red blood cells, the cells and the red blood cells are in extracellular fluid in the experiment cavity, electrode fluid is filled in a glass electrode, the micropipette is connected with the clamping end of the micropipette clamp holder, the micropipette clamp holder is arranged on a piezoelectric motion platform, the piezoelectric motion platform is arranged on a first three-dimensional micromanipulator, and the first three-dimensional micromanipulator is fixed on the experiment platform; the piezoelectric motion platform controls the red blood cells to execute repeated forward-backward motion circulation by controlling the micro-suction pipe to drive the red blood cells to move, namely the motion circulation of repeated contact-separation of the red blood cells and the cells, and records the adhesion state of the cells and the red blood cells in each contact-separation process through real-time images of a display.
The patch clamp comprises a patch clamp amplifier, a patch clamp probe and a patch clamp probe bracket, wherein the patch clamp probe is fixed on the experiment platform through the patch clamp probe bracket, a recording electrode of the patch clamp probe is connected to the tail end of a glass electrode holder through a BNC patch cord, the glass electrode is connected to the holding end of the glass electrode holder, and the glass electrode holder is arranged on a second three-dimensional micromanipulator; and the reference electrode is connected into extracellular fluid in the experiment cavity through a through hole on the side surface of the experiment cavity framework.
In the fluorescence imaging module, full spectrum light emitted by a light source of a bright field mercury lamp emits a bright field light path with a specific waveband through a bright field incident light color filter, an experiment cavity is irradiated under the light path, the bright field incident light color filter is arranged between the light source of the bright field mercury lamp and the experiment cavity, an experiment platform is arranged above an objective lens of an inverted microscope, the objective lens of the inverted microscope is right opposite to the center of the experiment cavity, and a fluorescence light path incident light dichroic spectroscope and a holophote are arranged inside the inverted microscope; the side surfaces of the upper part and the lower part of the inverted microscope are both provided with light path openings, the light path opening of the side surface of the lower part of the inverted microscope is connected with one end of a light splitter, a fluorescent light path emergent light dichromatic spectroscope is arranged in the light splitter, and two light path openings at the other end of the light splitter are respectively connected with and installed with a high-speed industrial camera and a fluorescent camera; a light beam emitted by a light source of a bright field mercury lamp is formed into a bright field light path after passing through a bright field incident light color filter to irradiate cells in the experimental cavity, and after being transmitted, the light beam sequentially passes through an objective lens at the top of the inverted microscope and a fluorescent light path incident light dichroic beam splitter, then is reflected to a fluorescent light path emergent light dichroic beam splitter inside the beam splitter through a holophote, then passes through the fluorescent light path emergent light dichroic beam splitter and is received by a high-speed industrial camera; an opening of a light path on the upper side surface of the inverted microscope is connected with an industrial camera, a bright field light path is used for splitting light inside the inverted microscope, wherein 20% of light beams are incident on the industrial camera through the opening of the light path, and the industrial camera is connected with a display; the full spectrum light emitted by the fluorescent light source is incident to a fluorescent incident light color filter in the inverted microscope through an upper side interface of the inverted microscope to form fluorescent incident light with a fixed waveband, then the fluorescent incident light is reflected to cells in the experimental cavity through a fluorescent light path incident light dichroic beam splitter, fluorescent emitted light emitted by fluorescent protein/fluorescent dye in the cells returns to the fluorescent light path incident light dichroic beam splitter through an objective lens of the inverted microscope to be transmitted, then the fluorescent light path emergent light dichroic beam splitter in the beam splitter is reflected through a holophote, and the fluorescent emitted light is received by a fluorescent camera after being reflected by the fluorescent light path emergent light dichroic beam splitter to form a fluorescent emergent light path.
The device can record the adhesion state of the cells and the red blood cells under the control of different membrane potentials, and simultaneously collect a fluorescence image with high signal-to-noise ratio to research the transmembrane signal conduction of the membrane protein.
The device also comprises a computer host, wherein the recording electrode is connected to the patch clamp amplifier through the glass electrode clamp and the BNC patch cord, the patch clamp amplifier is connected to the computer host through a USB interface, the piezoelectric motion platform is connected with the computer host through the piezoelectric motion platform controller, and the fluorescent light source and the fluorescent camera are directly connected with the computer host through the USB interface.
The specific implementation is to take the spectral characteristic detection of green fluorescent protein as an example, and the light source of the bright field mercury lamp emits full spectrum light; the wave band of the bright field incident light color filter is 617/73 nm; the wavelength band of the fluorescence incident light color filter is 465-495nm, the wavelength band of the fluorescence light path incident light dichroic beam splitter is 505nm (light with the wavelength less than 505nm is reflected, light with the wavelength more than 505 n'm is transmitted), and the wavelength band of the fluorescence light path emergent light dichroic beam splitter is 580nm (light with the wavelength less than 580nm is reflected, and light with the wavelength more than 580nm is transmitted).
The side of the experiment cavity framework is provided with a side through hole in advance, and a reference electrode of the patch clamp probe is arranged in the side through hole in a penetrating mode and is stably connected into extracellular fluid in the experiment cavity.
The invention integrates the fluorescence imaging technology, the patch clamp technology and the micro-suction tube. The integrated experimental control program comprises the necessary functions of a fluorescence imaging technology, a patch clamp technology and a micro-pipette, can record the coupling relation of three-spectrum phase information while synchronously recording the three-spectrum phase information, and can restore the corresponding relation of a collision state, a voltage control signal, a current sampling signal and a fluorescence spectrum sampling image controlled by the micro-pipette technology on the same time scale in an off-line manner. The integrated fluorescence-patch clamp-micropipette detection device can implement cooperative control on the components of a piezoelectric motion platform, a patch clamp amplifier, a fluorescence light source, a fluorescence camera and the like through a computer host.
The invention has the beneficial effects that:
the invention can detect the influence of the potential change of the cell membrane on the interaction of the membrane protein and simultaneously observe the influence of the potential change of the cell membrane on the transmembrane signal conduction of the membrane protein in real time. The invention can synchronously record three spectral phase information such as fluorescence spectrum, electrophysiological spectrum, adhesion state spectrum and the like and simultaneously record the coupling relation among the three spectral phase information.
The invention mainly aims at the influence of membrane potential change on the dynamic function of membrane protein and transmembrane signal conduction of the membrane protein in the field of life science. The invention has the following advantages:
1) the influence of the membrane potential change on the dynamic function of the membrane protein can be directly detected;
2) optical elements such as a color filter and the like in a fluorescence light path can be specifically changed according to the characteristics of the fluorescent protein/fluorescent dye, so that a fluorescence image with high signal-to-noise ratio is obtained to study transmembrane signal conduction of the membrane protein;
2) the coupling relation of the three-spectrum phase information can be recorded while the three-spectrum phase information is synchronously recorded, and the method can be used for restoring the corresponding relation of a collision state, a voltage control signal, a current sampling signal and a fluorescence spectrum sampling image controlled by the micropipette on the same time scale in an off-line manner.
Drawings
FIG. 1 is a system layout of the present invention.
FIG. 2 is an experimental schematic of the present invention.
Figure 3 is a design drawing of the experimental chamber skeleton according to the present invention.
In the figure: 1. a bright field mercury lamp light source, 2, a bright field incident light color filter, 3, a bright field light path, 4, a fluorescence light path incident light dichroic spectroscope, 5, an industrial camera for observing an adhesion state, 6, a display for observing an adhesion state, 7, a holophote, 8, a spectroscope, 9, a fluorescence light path emergent light dichroic spectroscope, 10, a high-speed industrial camera, 11, a fluorescence light source, 12, a full spectrum light emitted by the fluorescence light source 11, 13, a fluorescence incident light color filter, 14, a fluorescence incident light path, 15, a fluorescence emergent light path, 16, a fluorescence camera, 17, a computer host, 18, a piezoelectric motion platform controller, 19, a piezoelectric motion platform, 20, a patch clamp amplifier, 21, a micropipette holder, 22, an experimental cavity, 23, a first three-dimensional micromanipulator for controlling the micropipette, 24, a second three-dimensional micromanipulator for controlling a glass electrode, 25. glass electrode, 26, glass electrode holder, 27, BNC patch cord, 28, inverted microscope, 29, recording electrode, 30, electrode liquid, 31 experiment platform, 32, fluorescent cell in whole cell recording mode, 33, red blood cell, 34, micropipette, 35, chamber bottom glass sheet, 36, experiment chamber skeleton, 37 and side through hole.
Detailed Description
The invention is further illustrated by the following figures and examples.
As shown in fig. 1 and 2, the embodied device comprises a piezoelectric motion platform 19, a micropipette holder 21, an experiment cavity 22, a first three-dimensional micromanipulator 23, a second three-dimensional micromanipulator 24, a glass electrode 25, a recording electrode 29, an experiment platform 31, a micropipette 34 and an experiment cavity framework 36; the experiment platform 31 is provided with an experiment cavity 22, a first three-dimensional micromanipulator 23, a second three-dimensional micromanipulator 24, a patch clamp probe bracket and the like. As shown in fig. 3, the experiment cavity framework 36 is located in the center of the experiment platform 31, the upper and lower parallel glass sheets are adhered to the surface of the experiment cavity framework 36, the two glass sheets form the experiment cavity 22, and two sides of the experiment cavity 22 are hollowed out for the micro-suction tube 34 and the glass electrode 25 to enter;
the present invention redesigns the structure of the test chamber skeleton 36. Specifically, in order to make it easier to form a high-resistance seal to a cell, the invention increases the inclinable angle of the glass electrode 25 by increasing the thickness between the upper and lower glass sheets of the frame 36 of the experimental cavity. A side through hole 37 is reserved in the side of the experiment cavity framework 36, and a reference electrode on the patch clamp probe is connected into extracellular fluid in the experiment cavity through the side through hole 37.
A fluorescent cell 32 in a whole cell recording mode is arranged on the bottom surface 35 of the experiment cavity 22, a micro-pipette 34 sucks an erythrocyte 33, the cell 32 and the erythrocyte 33 are in extracellular fluid in the experiment cavity 22, electrode fluid 30 is filled in the glass electrode 25, the micro-pipette 34 is connected with the clamping end of a micro-pipette holder 21, the micro-pipette holder 21 is arranged on a piezoelectric motion platform 19, the piezoelectric motion platform 19 is arranged on a first three-dimensional micromanipulator 23, and the first three-dimensional micromanipulator 23 is fixed on the experiment platform 31; the piezoelectric motion platform 19 can control the micro-suction tube 34 to drive the red blood cell 33 to move, and control the red blood cell 33 to execute a repeated forward-backward movement cycle, i.e. a movement cycle of repeated contact-separation of the red blood cell 33 and the cell 32, and record the adhesion state of the cell 32 and the red blood cell 33 in each contact-separation process through a real-time image of the display 6.
The patch clamp related parts comprise a patch clamp amplifier 20, a patch clamp probe and a patch clamp probe bracket, wherein the patch clamp probe is fixed on an experiment platform 31 through the patch clamp probe bracket, a recording electrode of the patch clamp probe is connected to the tail end of a glass electrode holder 26 through a BNC patch cord 27, a glass electrode 25 is connected to the holding end of the glass electrode holder 26, the glass electrode holder 26 is installed on a second three-dimensional micromanipulator 24, and the second three-dimensional micromanipulator 24 is fixed on the experiment platform 31; the reference electrode is connected to the extracellular fluid in the experiment cavity through a through hole 37 on the side surface of the experiment cavity framework 36. The glass electrode 25 extends into the external electrode liquid in the experimental cavity, and the tail end of the recording electrode 29 is inserted into the internal electrode liquid 30 filled in the glass electrode 25
The first three-dimensional micromanipulator 23 and the second three-dimensional micromanipulator 24 are ultra-precise electric three-dimensional motion platforms which can be operated by a handle.
The fluorescence imaging module mainly comprises a bright field mercury lamp light source 1, a bright field incident light color filter 2, a bright field light path 3, a fluorescence light path incident light dichroic spectroscope 4, an industrial camera 5 for observing an adhesion state, a display 6 for observing an adhesion state, a total reflection mirror 7, a spectroscope 8, a fluorescence light path emergent light dichroic spectroscope 9, a high-speed industrial camera 10, a fluorescence light source 11, a full spectrum light 12 emitted by the fluorescence light source 11, a fluorescence incident light color filter 13, a fluorescence incident light path 14, a fluorescence emergent light path 15, a fluorescence camera 16 and an inverted microscope 28.
As shown in FIG. 1, the fluorescence imaging device of the invention has a fluorescence light source 11, a fluorescence camera 16 and other devices on fluorescence imaging, and optical devices such as a filter, a reflector, a dichroic beam splitter, a beam splitter and the like are added on the light path of an inverted microscope according to the excitation and emission spectrum of fluorescent protein/fluorescent dye, and the wave band of the fluorescence light path is separated from the wave band of bright field light to enhance the signal-to-noise ratio of the fluorescence image.
Specifically, full spectrum light emitted by a light field mercury lamp light source 1 emits a light field light path 3 with a specific waveband through a light field incident light color filter 2, an experiment cavity 22 is irradiated towards the lower part, the light field incident light color filter 2 is arranged between the light field mercury lamp light source 1 and the experiment cavity 22, an experiment platform 31 is installed above an objective lens of an inverted microscope 28, the objective lens of the inverted microscope 28 is opposite to the center of the experiment cavity 22, and a fluorescence light path incident light dichroic spectroscope 4 and a total reflector 7 are arranged inside the inverted microscope 28; the upper side and the lower side of the inverted microscope 28 are both provided with light path interfaces, the lower side interface of the inverted microscope 28 is connected with one end of the optical splitter 8, a fluorescence light path emergent light dichromatic spectroscope 9 is arranged in the optical splitter 8, and two interfaces at the other end of the optical splitter 8 are respectively connected with and installed with the high-speed industrial camera 10 and the fluorescence camera 16; a light beam emitted by a light source 1 of a bright field mercury lamp downwards and directly downwards passes through a bright field incident light color filter 2 to form a bright field light path 3 to irradiate cells in the experiment cavity 22, the transmitted light beam sequentially passes through an objective lens at the top of an inverted microscope 28 and a fluorescent light path incident light dichroic spectroscope 4, then is reflected to a fluorescent light path emergent light dichroic spectroscope 9 in a spectroscope 8 through a holophote 7, then passes through the fluorescent light path emergent light dichroic spectroscope 9 and is received by a high-speed industrial camera 10; the upper side interface of the inverted microscope 28 is connected with the industrial camera 5, the bright field light path 3 splits inside the inverted microscope 28, wherein 20% of light beams are incident on the industrial camera 5 through the interface, and the industrial camera 5 is connected with the display 6; the full spectrum light 12 emitted by the fluorescent light source 11 is incident to a fluorescent incident light color filter 13 inside the inverted microscope 28 through an upper side interface of the inverted microscope 28 to form fluorescent incident light 14 with a specified waveband, and then is reflected to the experiment cavity 22 through a fluorescent light path incident light dichroic beam splitter 4; the fluorescence emission light emitted by the fluorescent protein/fluorescent dye in the cell returns to the fluorescence light path incident light dichroic spectroscope 4 through the objective lens of the inverted microscope 28 to be transmitted, is reflected to the fluorescence light path emergent light dichroic spectroscope 9 in the spectroscope 8 through the total reflection mirror 7, is reflected by the fluorescence light path emergent light dichroic spectroscope 9 and then is received by the fluorescence camera 16 to form a fluorescence emergent light path 15;
the device can record the adhesion state of the cells and the red blood cells under the control of different membrane potentials, and simultaneously collect a fluorescence image with high signal-to-noise ratio to research the transmembrane signal conduction of the membrane protein.
The invention also comprises a computer host 17, a recording electrode 29 is connected to the patch clamp amplifier 20 through a glass electrode holder 26 and a BNC adapter wire 27, the patch clamp amplifier 20 is connected to the computer host 17 through a USB interface, the piezoelectric motion platform 19 is connected with the computer host 17 through a piezoelectric motion platform controller 18, and the fluorescent light source 11 and the fluorescent camera 16 are directly connected with the computer host 17 through the USB interface. The piezoelectric motion platform controller 18, the patch clamp amplifier 20, the fluorescent light source 11, the fluorescent camera 16 and other components are cooperatively controlled through the computer host 17.
Taking the spectral characteristic of green fluorescent protein as an example, the invention comprises the following steps:
the light source 1 of the bright field mercury lamp emits full spectrum light.
The wave band of the bright field incident light color filter 2 is 617/73 nm.
The wavelength band of the fluorescence incident light color filter 13 is 465-495nm, the wavelength band of the fluorescence light path incident light dichroic beam splitter 4 is 505nm, light with the wavelength less than 505nm is reflected, and light with the wavelength more than 505nm is transmitted; the wavelength band of the light emitted from the dichroic beam splitter 9 on the fluorescence light path is 580nm, light with a wavelength less than 580nm is reflected, and light with a wavelength greater than 580nm is transmitted.
A side through hole 37 is reserved in the side face of the experiment cavity framework 36, and a reference electrode of the patch clamp probe is stably connected into extracellular fluid in the experiment cavity 22 through the side through hole 37.
The specific implementation working process of the invention is as follows:
the invention designs a new experimental scheme based on the experimental purpose of a fluorescence-patch clamp-micropipette detection device.
Specifically, in the preparation process of the experimental cavity 22, the suspension cells 32 expressing the target membrane protein molecules and intracellular fluorescent proteins are adhered to the glass sheet 35 on the bottom surface of the experimental cavity through polylysine and are in a semi-adherent state;
the red blood cells 33 with the surface connected with another protein molecule are added into the extracellular fluid in the experimental cavity 22;
an experimental operator selects a target cell 32 with a smooth surface and a strong fluorescence signal, moves the target cell 32 to the middle of a visual field by moving an objective table of the inverted microscope 28, and controls the glass electrode 25 to perform the operations of sealing, membrane rupture, compensation and the like on the target cell 32 by operating the second three-dimensional micromanipulator 24;
the experiment operator controls the micro-suction pipe 34 to suck a red blood cell 33 by operating the first three-dimensional micromanipulator 23;
setting voltage stimulation parameters (waveform, amplitude, frequency and the like), micropipette experiment parameters (contact time of the red blood cells 33 and the cells 32 and the like), and fluorescence imaging module sampling parameters (exposure time, sampling interval and the like) by an experiment operator;
starting a data acquisition stage: the program automatically applies set voltage stimulation to the cells 32 in the whole-cell recording mode, and the program automatically displays and records current voltage and current information; the program cooperatively controls the fluorescent light source 11 and the fluorescent camera 16, automatic sampling is carried out according to set fluorescent parameters (exposure time, sampling interval and the like), and after each sampling is finished, images of a fluorescent spectrum are automatically stored as independent files in a hard disk of the host 17 in an intensity array form; the program controls the red blood cells 33 to perform a cycle of movement of repeated contact-separation with the cells 32, and the experiment operator records the adhesion state of the cells 32 and the red blood cells 33 during each contact-separation process through real-time images of the display 6.
After the recording is finished (the recording time is the time of 50 contact-separation movement cycles), the voltage and current data in the current recording process and the adhesion state recorded manually (the frequency of the adhesion state is recorded as the adhesion frequency under the current membrane potential stimulation condition) are saved.
The experiment operator modifies the voltage stimulation parameters according to the experiment design and repeats the data acquisition stage;
by comparing the change of adhesion frequency under different voltage stimulation and the change of fluorescence signals in cells along with time, the micropipette technology of the integrated patch clamp can analyze the influence of the change of membrane potential on the interaction of membrane proteins and the influence of the change of the membrane potential on the transmembrane signal conduction of the membrane proteins.
In particular, the invention can record the coupling relation of three-spectrum information at the same time of synchronously recording three-spectrum information such as fluorescence spectrum, electrophysiological spectrum, adhesion state spectrum and the like. Specifically, at the same time when the program starts the data acquisition phase, the fluorescence camera acquires a first image (at this time, the adhesion state spectrum controls the red blood cells 33 to start moving, and the electrophysiological spectrum starts outputting a voltage control signal), and then samples are performed at fixed time intervals; after that, at the moment when the movement state of the piezoelectric movement platform 19 changes (starts or stops moving), the data index of the electrophysiological spectrum is recorded into an independent index array file, and the index array file can be used for off-line restoration of the corresponding relationship between the movement state of the red blood cell 33 controlled by the micropipette technology and the voltage control signal and the current sampling signal on the same time scale. Therefore, the information of the three spectral phases can be corresponded on the same time scale, and the coupling relation of the information of the three spectral phases can be analyzed.
The foregoing detailed description is intended to illustrate and not limit the invention, which is intended to be within the spirit and scope of the appended claims, and any changes and modifications that fall within the true spirit and scope of the invention are intended to be covered by the following claims.
Claims (4)
1. The utility model provides a fluorescence-patch clamp-micropipette detection device which characterized in that: the device comprises an experiment platform (31), a bright-field mercury lamp light source (1), an inverted microscope (28), a glass electrode (25), a micropipette (34), a piezoelectric motion platform (19), a first three-dimensional micromanipulator (23), a recording electrode (29) and an experiment cavity framework (36); an experiment cavity (22), a first three-dimensional micromanipulator (23), a second three-dimensional micromanipulator (24) and a patch clamp are arranged on the experiment platform (31), an experiment cavity framework (36) is located in the center of the experiment platform (31), an upper glass sheet and a lower glass sheet which are parallel are placed on the experiment cavity framework (36), the experiment cavity (22) is formed between the two glass sheets, and two sides of the experiment cavity (22) are hollowed out for a micropipette (34) and a glass electrode (25) to enter; fluorescent cells (32) in a whole cell recording mode are arranged on the bottom surface (35) of the experiment cavity (22), a micropipette (34) absorbs red blood cells (33), the cells (32) and the red blood cells (33) are in extracellular fluid in the experiment cavity (22), electrode fluid (30) is filled in a glass electrode (25), the micropipette (34) is connected with the clamping end of a micropipette clamp holder (21), the micropipette clamp holder (21) is installed on a piezoelectric motion platform (19), the piezoelectric motion platform (19) is installed on a first three-dimensional micromanipulator (23), and the first three-dimensional micromanipulator (23) is fixed on the experiment platform (31); the patch clamp comprises a patch clamp amplifier (20), a patch clamp probe and a patch clamp probe bracket, wherein the patch clamp probe is fixed on an experiment platform (31) through the patch clamp probe bracket, a recording electrode of the patch clamp probe is connected to the tail end of a glass electrode holder (26) through a BNC (bayonet nut connector) patch cord (27), a glass electrode (25) is connected to the holding end of the glass electrode holder (26), and the glass electrode holder (26) is arranged on a second three-dimensional micromanipulator (24); the reference electrode is connected into extracellular fluid in the experimental cavity;
in the fluorescence imaging module, full spectrum light emitted by a bright field mercury lamp light source (1) emits a bright field light path (3) with a specific waveband through a bright field incident light color filter (2), an experiment cavity (22) is illuminated downwards and directly below, the bright field incident light color filter (2) is arranged between the bright field mercury lamp light source (1) and the experiment cavity (22), an experiment platform (31) is installed above an objective lens of an inverted microscope (28), the objective lens of the inverted microscope (28) is right opposite to the center of the experiment cavity (22), and a fluorescence light path incident light dichroic spectroscope (4) and a total reflector (7) are arranged inside the inverted microscope (28); light path openings are formed in the upper side face and the lower side face of the inverted microscope (28), the light path opening in the lower side face of the inverted microscope (28) is connected with one end of the light splitter (8), a fluorescent light path emergent light dichroic light splitter (9) is arranged in the light splitter (8), and two light path openings in the other end of the light splitter (8) are respectively connected with and mounted with the high-speed industrial camera (10) and the fluorescent camera (16); a light beam emitted by a light field mercury lamp light source (1) downwards and directly downwards passes through a light field incident light color filter (2) to form a light field light path (3) to irradiate cells in an experimental cavity (22), after being transmitted, the light beam sequentially passes through an objective lens at the top of an inverted microscope (28) and a fluorescent light path incident light dichroic spectroscope (4), then is reflected to a fluorescent light path emergent light dichroic spectroscope (9) in a light splitter (8) through a total reflector (7), then passes through the fluorescent light path emergent light dichroic spectroscope (9), and is received by a high-speed industrial camera (10); an opening of an optical path on the upper side surface of the inverted microscope (28) is connected with the industrial camera (5), a bright field optical path (3) is split inside the inverted microscope (28), a light beam is incident on the industrial camera (5) through the opening of the optical path, and the industrial camera (5) is connected with the display (6); the full spectrum light (12) emitted by a fluorescent light source (11) is incident to a fluorescent incident light color filter (13) in an inverted microscope (28) through an upper side interface of the inverted microscope (28) to form fluorescent incident light (14) with a fixed waveband, then the fluorescent incident light is reflected to cells in an experimental cavity (22) through a fluorescent light path incident light dichroic spectroscope (4), fluorescent emitted light emitted by fluorescent protein/fluorescent dye in the cells returns to the fluorescent light path incident light dichroic spectroscope (4) through an objective lens of the inverted microscope (28) to be transmitted, then the fluorescent emitted light is reflected to a fluorescent emergent light path dichroic spectroscope (9) in a spectroscope (8) through a total reflection mirror (7), and the fluorescent emergent light is received by a fluorescent camera (16) after being reflected by the fluorescent light path emergent light dichroic spectroscope (9) to form a fluorescent emergent light.
2. The fluorescence-patch clamp-micropipette assay device of claim 1, wherein: the device is characterized by further comprising a computer host (17), a recording electrode (29) is connected to a patch clamp amplifier (20) through a glass electrode clamp (26) and a BNC patch cord (27), the patch clamp amplifier (20) is connected to the computer host (17), a piezoelectric motion platform (19) is connected with the computer host (17) through a piezoelectric motion platform controller (18), and a fluorescent light source (11) and a fluorescent camera (16) are directly connected with the computer host (17).
3. The fluorescence-patch clamp-micropipette assay device of claim 1, wherein: the specific implementation is to take the spectral characteristic detection of green fluorescent protein as an example, and the light source (1) of the bright field mercury lamp emits full spectrum light; the wave band of the bright field incident light color filter (2) is 617/73 nm; the wave band of the fluorescence incident light color filter (13) is 465-495nm, the wave band of the fluorescence light path incident light dichroic spectroscope (4) is 505nm, and the wave band of the fluorescence light path emergent light dichroic spectroscope (9) is 580 nm.
4. The fluorescence-patch clamp-micropipette assay device of claim 1, wherein: a side through hole (37) is reserved in the side of the experiment cavity framework (36), and a reference electrode of the patch clamp probe penetrates through the side through hole (37) and is stably connected into extracellular fluid in the experiment cavity (22).
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