WO2021114790A1 - 一种荧光—膜片钳—微吸管检测装置 - Google Patents

一种荧光—膜片钳—微吸管检测装置 Download PDF

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WO2021114790A1
WO2021114790A1 PCT/CN2020/115249 CN2020115249W WO2021114790A1 WO 2021114790 A1 WO2021114790 A1 WO 2021114790A1 CN 2020115249 W CN2020115249 W CN 2020115249W WO 2021114790 A1 WO2021114790 A1 WO 2021114790A1
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fluorescent
light
patch clamp
micropipette
light path
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PCT/CN2020/115249
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English (en)
French (fr)
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陈伟
安宸毅
刘俊伟
胡炜
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浙江大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells

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  • the invention relates to the integration of fluorescence imaging technology, patch clamp technology and micropipette technology, in particular to a detection device capable of studying membrane protein transmembrane signal conduction while detecting the regulation law of membrane potential on the interaction between protein molecules.
  • the membrane potential of the cell membrane is a key regulatory factor for cell life activities.
  • the membrane potential of neurons plays an important role in regulating the various life activities of neurons, and is an important biophysical factor that dynamically regulates the structure and function of the brain neural network; in non-neuronal cells, the membrane potential of the cell also regulates its proliferation , Differentiation and other life activities.
  • Patch clamp technology is an effective method to study membrane potential related issues, and its whole-cell recording mode can achieve accurate and rapid control of the membrane potential of the entire cell membrane.
  • patch clamp is currently mainly limited to the study of ion channel characteristics.
  • Membrane proteins are the main sensors for cells to perceive the external environment and respond. How most protein molecules on the cell membrane sense changes in membrane potential to adjust their dynamic functions has not yet been resolved. The main bottleneck is the lack of direct and effective research methods.
  • Micropipette technology is widely used in in-situ detection of protein-protein interaction kinetic parameters.
  • the protein molecules are always in the cell membrane microenvironment (the protein molecules to be tested are connected and expressed on the red blood cells and the cell surface respectively). Therefore, the micropipette experiment has a unique advantage in detecting the interaction between membrane receptors and ligands.
  • the fusion of patch clamp technology and micropipette technology provides the possibility to study the dynamic regulation of membrane potential changes on the interaction between membrane proteins under physiological conditions.
  • fluorescence imaging technology is widely used in the study of membrane protein signal transduction. Only integrating patch clamp technology into micropipette technology can only study the effect of membrane potential changes on membrane protein interactions, but cannot study its effect on membrane proteins. The influence of transmembrane signals. Therefore, based on the integration of patch clamp technology, the further integration of fluorescence imaging technology can further study the transmembrane signal transduction of membrane proteins. Fluorescence imaging-patch clamp-micropipette technology, which integrates the three spectral phase information of fluorescence spectrum, electrophysiological spectrum, and adhesion frequency, can detect the effect of cell membrane potential changes on membrane protein interactions and observe its impact on membrane protein in real time. The influence of membrane signaling.
  • the purpose of the present invention is to provide a fluorescence-patch clamp-micropipette detection device that can detect the effect of changes in cell membrane potential on membrane protein interactions while simultaneously observing its effect on membrane proteins in real time.
  • the influence of transmembrane signaling can simultaneously record the three-spectral phase information such as the fluorescence spectrum, the electrophysiological spectrum and the adhesion state spectrum, and simultaneously record the coupling relationship between the three-spectral phase information.
  • the invention includes an experimental platform, a bright-field mercury lamp light source, an inverted microscope, a glass electrode, a micro-pipette, a piezoelectric motion module, a first three-dimensional micromanipulator, a recording electrode and an experimental cavity skeleton.
  • the experimental platform is equipped with an experimental cavity, the first three-dimensional micromanipulator, the first three-dimensional micromanipulator, the patch clamp probe holder, etc.
  • the experimental cavity skeleton is located in the center of the experimental platform, and the experimental cavity skeleton is placed with two upper and lower pieces.
  • the two glass slides form an experimental chamber between the two glass slides.
  • the two sides of the experimental chamber are hollowed out for micropipettes and glass electrodes to enter; the bottom surface of the experimental chamber has fluorescent cells in the whole-cell recording mode, and the micropipette Aspirate red blood cells.
  • the cells and red blood cells are in the extracellular fluid in the experimental cavity.
  • the glass electrode is filled with the electrode fluid.
  • the micropipette is connected to the clamping end of the micropipette holder, and the micropipette holder is installed on the piezoelectric motion platform.
  • the piezoelectric motion platform is installed on the first three-dimensional micromanipulator, and the first three-dimensional micromanipulator is fixed on the experimental platform; the piezoelectric motion platform drives the movement of red blood cells by controlling the micropipette, and controls the red blood cells to perform repeated advancement—
  • the retreat movement cycle is the movement cycle of repeated contact-separation between red blood cells and cells, and the adhesion state of cells and red blood cells during each contact-separation process is recorded through the real-time image of the monitor.
  • the patch clamp includes a patch clamp amplifier, a patch clamp probe, and a patch clamp probe holder.
  • the patch clamp probe is fixed on the experimental platform through the patch clamp probe holder.
  • the recording electrode of the patch clamp probe is connected to the glass through a BNC adapter cable.
  • the glass electrode is connected to the clamping end of the glass electrode holder, and the glass electrode holder is installed on the second three-dimensional micromanipulator; the reference electrode is connected to the through hole on the side of the experimental cavity skeleton In the extracellular fluid in the experimental cavity.
  • the full-spectrum light emitted by the brightfield mercury lamp light source emits a specific wavelength of brightfield light path through the brightfield incident light filter, and illuminates the experimental cavity directly below, which is one of the brightfield mercury lamp light source and the experimental cavity.
  • Bright field incident light filter is arranged in the room, the experiment platform is installed above the objective lens of the inverted microscope, the objective lens of the inverted microscope is facing the center of the experiment cavity, and the inside of the inverted microscope is equipped with a fluorescent light path incident light dichroic mirror and a total reflection mirror; inverted microscope Both the upper and lower sides are provided with optical path openings. The lower side of the inverted microscope is connected to one end of the spectroscope.
  • the spectroscope is equipped with a fluorescent light path outgoing light dichroic spectroscope.
  • the two optical path openings at the other end of the spectroscope are respectively connected to install high-speed industrial Camera and fluorescent camera; bright-field mercury lamp light source facing downwards emits a light beam directly below the bright-field incident light filter to form a bright-field light path to illuminate the cells in the experimental cavity, and then pass through the objective lens on the top of the inverted microscope and the fluorescent light path in turn
  • the light dichroic beam splitter is reflected by the total reflection mirror to the fluorescent light path inside the beam splitter, the emitted light dichroic beam splitter, and then transmitted through the fluorescent light path, the emitted light dichroic beam splitter is then received by the high-speed industrial camera;
  • the upper side optical path opening of the inverted microscope is connected to the industry Camera, the bright field light path is split inside the inverted microscope, 20% of the light beam is incident on the industrial camera through the light path opening, and the industrial camera is connected to the
  • the invention can record the adhesion state of cells and red blood cells under the control of different membrane potentials through the above-mentioned device, and at the same time collect fluorescence images with high signal-to-noise ratio to study the transmembrane signal conduction of membrane proteins.
  • the recording electrode is connected to the patch clamp amplifier via a glass electrode holder and a BNC adapter cable.
  • the patch clamp amplifier is connected to the host computer via a USB interface.
  • the piezoelectric motion platform is connected to the computer via the piezoelectric motion platform controller. Host connection, fluorescent light source and fluorescent camera are directly connected to the computer host through the USB interface.
  • the specific implementation takes the detection of the spectral characteristics of green fluorescent protein as an example.
  • the bright-field mercury lamp light source emits full-spectrum light; the bright-field incident light filter has a wavelength band of 617/73 nm; and the fluorescent incident light filter is
  • the wavelength of the plate is 465-495nm, the wavelength of the incident light of the fluorescent light path is 505nm (light with a wavelength less than 505nm is reflected, and light with a wavelength greater than 505n'm is transmitted), and the wavelength of the light emitted from the fluorescent 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 is reserved on the side of the frame of the experiment cavity, and the reference electrode of the patch clamp probe is arranged through the side through hole, and is stably connected to the extracellular fluid in the experiment cavity.
  • the invention integrates fluorescence imaging technology, patch clamp technology, and micropipette.
  • the integrated experimental control program contains the necessary functions of fluorescence imaging technology, patch clamp technology, and micropipette. It can record the coupling relationship of the three spectrum information while simultaneously recording the trispectral information, and can restore the control of the micropipette technology offline Correspondence between the collision state, voltage control signal, current sampling signal, and fluorescence spectrum sampling image on the same time scale.
  • the integrated fluorescence-patch clamp-micropipette detection device can coordinately control components such as the piezoelectric motion platform, patch clamp amplifier, fluorescent light source, fluorescent camera, etc. through the host computer.
  • the present invention can detect the influence of cell membrane potential change on membrane protein interaction while observing its influence on membrane protein transmembrane signal conduction in real time.
  • the invention can simultaneously record the three-spectral phase information such as the fluorescence spectrum, the electrophysiological spectrum and the adhesion state spectrum, and simultaneously record the coupling relationship between the three-spectral phase information.
  • the present invention is mainly aimed at the influence of membrane potential changes on the dynamic functions of membrane proteins in the field of life sciences and the transmembrane signal transduction of membrane proteins.
  • the present invention has the following advantages:
  • the optical components such as filters in the fluorescent light path can be specifically changed to obtain fluorescent images with high signal-to-noise ratio to study the transmembrane signal transduction of membrane proteins;
  • Figure 1 is a system design diagram of the present invention.
  • Figure 2 is a schematic diagram of the experiment of the present invention.
  • Fig. 3 is a design diagram of the experimental cavity skeleton involved in the present invention.
  • the specific implementation device includes a piezoelectric motion platform 19, a micropipette holder 21, an experimental cavity 22, a first three-dimensional micromanipulator 23, a second three-dimensional micromanipulator 24, and glass Electrode 25, recording electrode 29, experimental platform 31, micropipette 34, and experimental cavity skeleton 36; the experimental platform 31 is arranged with an experimental cavity 22, a first three-dimensional micromanipulator 23, and a second three-dimensional micromanipulator 24, Patch clamp probe holder, etc. As shown in Fig. 3, the experimental cavity skeleton 36 is located in the center of the experimental platform 31. Two parallel glass sheets are pasted on the surface of the experimental cavity skeleton 36. The two glass sheets form the experimental cavity 22, and the two sides of the experimental cavity 22 Hollow out for the micropipette 34 and glass electrode 25 to enter;
  • the structure of the experimental cavity skeleton 36 is redesigned. Specifically, in order to more easily form a high-resistance seal to the cells, the present invention increases the tiltable angle of the glass electrode 25 by increasing the thickness between the upper and lower glass sheets of the experimental cavity skeleton 36.
  • a side through hole 37 is reserved on the side of the experimental cavity skeleton 36, and the reference electrode on the patch clamp probe is connected to the extracellular fluid in the experimental cavity through the side through hole 37.
  • the bottom surface 35 of the experimental cavity 22 has fluorescent cells 32 in the whole-cell recording mode.
  • the micropipette 34 sucks red blood cells 33.
  • the cells 32 and red blood cells 33 are in the extracellular fluid in the experimental cavity 22.
  • the glass electrode 25 is filled with The electrode inner fluid 30 and the micropipette 34 are connected to the clamping end of the micropipette holder 21, the micropipette holder 21 is installed on the piezoelectric motion platform 19, and the piezoelectric motion platform 19 is installed on the first three-dimensional micromanipulator 23 Above, the first three-dimensional micromanipulator 23 is fixed on the experimental platform 31; the piezoelectric motion platform 19 can drive the movement of the red blood cells 33 by controlling the micropipette 34, and control the red blood cells 33 to perform a repeated forward-backward movement cycle, that is, the red blood cells 33
  • the patch clamp related parts include the patch clamp amplifier 20, the patch clamp probe, and the patch clamp probe holder.
  • the patch clamp probe is fixed on the experimental platform 31 through the patch clamp probe holder.
  • the recording electrode of the patch clamp probe is transferred by BNC.
  • the wiring 27 is connected to the end of the glass electrode holder 26, and the glass electrode 25 is connected to the holding end of the glass electrode holder 26.
  • the glass electrode holder 26 is installed on the second three-dimensional micromanipulator 24.
  • the micromanipulator 24 is fixed on the experiment platform 31; the reference electrode is connected to the extracellular fluid in the experiment cavity through the through hole 37 on the side of the experiment cavity skeleton 36.
  • the glass electrode 25 extends into the electrode outer liquid in the experiment chamber, and the end of the recording electrode 29 is inserted into the electrode inner liquid 30 poured into the glass electrode 25
  • Both the first three-dimensional micromanipulator 23 and the second three-dimensional micromanipulator 24 are ultra-precision electric three-dimensional motion platforms that can be operated by a handle.
  • the fluorescence imaging module mainly includes bright field mercury lamp light source 1, bright field incident light color filter 2, bright field light path 3, fluorescent light path incident light dichroic spectroscope 4, industrial camera 5 for observing the adhesion state, Monitor the display 6, total reflection mirror 7, beam splitter 8, fluorescent light path dichroic beam splitter 9, high-speed industrial camera 10, fluorescent light source 11, full spectrum light 12 emitted by fluorescent light source 11, fluorescent incident light filter Color plate 13, fluorescence incident light path 14, fluorescence emission light path 15, fluorescence camera 16 and inverted microscope 28.
  • the present invention has a fluorescent light source 11, a fluorescent camera 16 and other equipment for fluorescence imaging, and adds a filter, a mirror, and a second mirror to the light path of the inverted microscope according to the excitation and emission spectra of the fluorescent protein/fluorescent dye.
  • Optical devices such as color splitters and beam splitters separate the wavelength band of the fluorescent light path from the bright field light band to enhance the signal-to-noise ratio of the fluorescent image.
  • the full-spectrum light emitted by the brightfield mercury lamp light source 1 emits a specific wavelength of the brightfield light path 3 through the brightfield incident light filter 2, and irradiates the experiment cavity 22 directly below, the brightfield mercury lamp light source 1 and the experiment
  • the bright field incident light filter 2 is arranged between the cavity 22, the experiment platform 31 is installed above the objective lens of the inverted microscope 28, the objective lens of the inverted microscope 28 is facing the center of the experiment cavity 22, and the inside of the inverted microscope 28 is provided with a fluorescent light path.
  • Color splitter 4 and total reflection mirror 7; both the upper and lower sides of the inverted microscope 28 are equipped with optical path interfaces, and the lower side of the inverted microscope 28 is connected to one end of the spectroscope 8.
  • the spectroscope 8 is equipped with a fluorescent light path outgoing light dichroic spectroscope 9.
  • the two ports at the other end of the beam splitter 8 are respectively connected to install high-speed industrial cameras 10 and fluorescent cameras 16; bright-field mercury lamp light source 1 emits light directly below the bright-field incident light filter 2 to form a bright-field optical path 3
  • the cells in the experimental cavity 22 are irradiated, and then transmitted through the objective lens on the top of the inverted microscope 28, the fluorescent light path, the incident light dichroic dichroic mirror 4, and the total reflection mirror 7 is reflected to the fluorescent light path inside the beam splitter 8 and the outgoing light dichroic dichroic dichroic mirror 9 , And then transmitted through the fluorescent light path, the dichroic dichroic mirror 9 is then received by the high-speed industrial camera 10; the upper side interface of the inverted microscope 28 is connected to the industrial camera 5, and the bright field light path 3 is split inside the inverted microscope 28, and 20% of the light beams pass through The interface is incident on the industrial camera
  • the full-spectrum light 12 emitted by the fluorescent light source 11 enters the fluorescent incident light filter 13 inside the inverted microscope 28 through the upper side interface of the inverted microscope 28 to form a specified wavelength band Fluorescent incident light 14, and then through the fluorescent light path, the incident light dichroic dichroic mirror 4 is reflected into the experimental cavity 22; the fluorescent emission light emitted by the fluorescent protein/fluorescent dye in the cell passes through the objective lens of the inverted microscope 28 and returns to the fluorescent light path.
  • the incident light dichroic The beam splitter 4 is transmitted, and then reflected by the total reflection mirror 7 to the fluorescent light path inside the beam splitter 8 to the dichroic beam splitter 9, and the light from the fluorescent light path is reflected by the dichroic beam splitter 9 and then received by the fluorescence camera 16 to form fluorescence.
  • the invention can record the adhesion state of cells and red blood cells under the control of different membrane potentials through the above-mentioned device, and at the same time collect fluorescence images with high signal-to-noise ratio to study the transmembrane signal conduction of membrane proteins.
  • the present invention also includes a computer host 17, the recording electrode 29 is connected to the patch clamp amplifier 20 via a glass electrode holder 26 and a BNC adapter cable 27, the patch clamp amplifier 20 is connected to the host computer 17 via a USB interface, and a piezoelectric motion platform 19 is connected to the host computer 17 via the piezoelectric motion platform controller 18, and the fluorescent light source 11 and the fluorescence camera 16 are directly connected to the host computer 17 through a USB interface.
  • the host computer 17 implements coordinated control of components such as the piezoelectric motion platform controller 18, the patch clamp amplifier 20, the fluorescent light source 11, and the fluorescent camera 16.
  • the present invention Taking the spectral characteristics of green fluorescent protein as an example, the present invention:
  • the bright-field mercury lamp light source 1 emits full-spectrum light.
  • the wavelength band of the bright-field incident light color filter 2 is 617/73nm.
  • the wavelength band of the fluorescent incident light filter 13 is 465-495nm
  • the wavelength band of the fluorescent light path incident light dichroic spectroscope 4 is 505nm, light with a wavelength less than 505nm is reflected, and light with a wavelength greater than 505nm is transmitted
  • the wavelength band of the color splitter 9 is 580 nm, and light with a wavelength less than 580 nm is reflected, and light with a wavelength greater than 580 nm is transmitted.
  • a side through hole 37 is reserved on the side of the experimental cavity skeleton 36 of the present invention, and the reference electrode of the patch clamp probe is stably connected to the extracellular fluid in the experimental cavity 22 through the side through hole 37.
  • the present invention designs a new experimental scheme based on the experimental purpose of the fluorescence-patch clamp-micropipette detection device.
  • the suspension cells 32 expressing the target membrane protein molecules and the intracellular fluorescent protein are adhered to the glass plate 35 on the bottom surface of the experimental cavity through polylysine, and are semi-adherent. status;
  • Red blood cells 33 with another protein molecule attached to the surface are added to the extracellular fluid in the experimental cavity 22;
  • the experiment operator selects the target cell 32 with smooth surface and strong fluorescence signal, and moves it to the middle of the field of view by moving the stage of the inverted microscope 28, and then controls the glass electrode 25 to the target by manipulating the second three-dimensional micromanipulator 24
  • Cell 32 performs routine whole-cell recording patch clamp operations such as sealing, rupture, and compensation;
  • the experiment operator controls the micropipette 34 to suck a red blood cell 33 by manipulating the first three-dimensional micromanipulator 23;
  • the experiment operator sets the voltage stimulation parameters (waveform, amplitude, frequency, etc.), the micropipette experiment parameters (the contact time between the red blood cells 33 and the cells 32, etc.), and the fluorescence imaging module sampling parameters (exposure time, sampling interval, etc.);
  • the program automatically applies the set voltage stimulation to the cells 32 in the whole cell recording mode, the program automatically displays and records the current voltage and current information; the program cooperates to control the fluorescent light source 11 and the fluorescent camera 16, according to the set Fluorescence parameters (exposure time, sampling interval, etc.) are automatically sampled. After each sampling, the image of the fluorescence spectrum is automatically stored as an independent file in the hard disk of the host 17 in the form of an intensity array; the program controls the execution of red blood cells 33 and repeats of cells 32 In the contact-separation movement cycle, the experimental operator records the adhesion state of the cells 32 and the red blood cells 33 through the real-time image of the display 6 during each contact-separation process.
  • the recording time is 50 contact-separation movement cycles
  • the voltage and current data during the recording process as well as the adhesion state recorded manually (the frequency of the adhesion state is recorded as the current membrane potential stimulation Adhesion frequency under conditions).
  • the experiment operator then modified the voltage stimulation parameters according to the experimental design, and repeated the data acquisition phase;
  • this integrated patch clamp micropipette technology can analyze the influence of membrane potential changes on membrane protein interactions and their effects on membrane proteins. The influence of transmembrane signaling.
  • the present invention can record the coupling relationship of the three-spectral phase information while simultaneously recording the three-spectral phase information such as the fluorescence spectrum, the electrophysiological spectrum, and the adhesion state spectrum.
  • the fluorescence camera collects the first image (at this time, the adhesion state spectrum controls the red blood cells 33 to start moving, and the electrophysiology spectrum starts to output voltage control signals), and then samples are taken at a fixed time interval; Thereafter, every time the motion state of the piezoelectric motion platform 19 changes (start motion or stop motion), the data index of the electrophysiological spectrum will be recorded in an independent index array file, which can be used to restore the micropipette offline
  • the corresponding relationship between the motion state of the red blood cell 33 controlled by the technology and the voltage control signal and the current sampling signal on the same time scale. In this way, the information of the three spectral phases can be corresponded on the same time scale, and the coupling relationship of the three spectral phase information can also be analyzed.

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Abstract

一种荧光—膜片钳—微吸管检测装置。实验平台(31)上布置有实验容腔(22)、三维显微操作器(23,24)、膜片钳，实验容腔(22)两侧镂空供微吸管(34)和玻璃电极(25)进入；实验容腔(22)有荧光细胞(32)，微吸管(34)吸取红细胞(33)，细胞(32)和红细胞(33)处于实验容腔(22)内的细胞外液中，玻璃电极(25)内充有电极内液(30)；膜片钳包括膜片钳放大器(20)、膜片钳探头、膜片钳探头支架；荧光光源(11)发射的全谱光(12)经滤色片(13)形成荧光入射光(14)，入射到实验容腔(22)中的细胞(32)，细胞(32)的荧光返回被荧光相机(16)接收。检测装置能够在检测细胞膜电位变化对膜蛋白相互作用的影响的同时采集高信噪比的荧光图像来研究膜蛋白的跨膜信号传导，能够在同步记录荧光谱、电生理谱、粘附状态谱等三个谱相信息的同时记录三谱相信息间的耦合关系。

Description

一种荧光—膜片钳—微吸管检测装置 技术领域
本发明涉及荧光成像技术、膜片钳技术、微吸管技术的整合，尤其是涉及了一种能够在检测膜电位对蛋白质分子间相互作用调控规律的同时研究膜蛋白跨膜信号传导的检测装置。
背景技术
细胞膜的膜电位是细胞生命活动的关键调控因素。神经元的膜电位对神经元的各项生命活动起到重要的调控作用，是动态调控脑神经网络结构与功能的重要生物物理因素；在非神经元细胞中，细胞的膜电位同样调控其增殖、分化等生命活动。膜片钳技术是研究膜电位相关问题的有效手段，其全细胞记录模式能够实现对整个细胞膜的膜电位实施准确而快速的控制。然而，膜片钳目前主要局限于离子通道特性的研究。
膜蛋白是细胞感知外界环境并作出响应的主要感受器，细胞膜上多数蛋白质分子如何感受膜电位变化来调整其动态功能尚未被解析。其主要瓶颈在于缺乏直接有效的研究手段。
微吸管技术被广泛应用于蛋白质－蛋白质相互作用动力学参数的原位检测。在微吸管实验的检测过程中，蛋白质分子时刻处于细胞膜微环境中(待测蛋白制分子分别被连接、表达在红细胞、细胞表面)。因此微吸管实验在检测膜受体、配体之间的相互作用时具有得天独厚的优势。膜片钳技术与微吸管技术两种技术的融合为研究生理条件下膜电位变化对膜蛋白之间相互作用的动态调控提供了可能。
此外，荧光成像技术被广泛应用于膜蛋白跨膜信号传导的研究，仅仅将膜片钳技术整合到微吸管技术中仅仅能够研究膜电位变化对膜蛋白相互作用的影响，无法研究其对膜蛋白跨膜信号的影响。因此，在膜片钳技术整合的基础上进一步整合荧光成像技术可以进一步研究膜蛋白的跨膜信号传导。整合了荧光谱、电生理谱、粘附频率三个谱相信息的荧光成像—膜片钳—微吸管技术能够在检测细胞膜电位变化对膜蛋白相互作用的影响的同时实时观测其对膜蛋白跨膜信号传导的影响。
发明内容
为了解决背景技术中提到的技术问题，本发明目的在于提供一种荧光—膜 片钳—微吸管检测装置，能够在检测细胞膜电位变化对膜蛋白相互作用的影响的同时实时观测其对膜蛋白跨膜信号传导的影响。本发明能够在同步记录荧光谱、电生理谱、粘附状态谱等三个谱相信息的同时记录三谱相信息间的耦合关系。
为实现上述发明目的，本发明采用的技术方案是：
本发明包括实验平台、明场汞灯光源、倒置显微镜、玻璃电极、微吸管、压电运动模块、第一三维显微操作器、记录电极和实验容腔骨架。
实验平台上布置有实验容腔、第一三维显微操作器、第一三维显微操作器、膜片钳探头支架等，实验容腔骨架位于实验平台中央，实验容腔骨架放置有上下两片平行的玻璃片，两片玻璃片中间形成实验容腔，实验容腔的两侧镂空，供微吸管和玻璃电极进入；实验容腔的底面上有处于全细胞记录模式下的荧光细胞，微吸管吸取红细胞，细胞和红细胞处于实验容腔内的细胞外液中，玻璃电极内充有电极内液，微吸管连接微吸管夹持器的夹持端，微吸管夹持器安装在压电运动平台上，压电运动平台安装在第一三维显微操作器上，第一三维显微操作器固定于实验平台上；压电运动平台通过控制微吸管带动红细胞的运动，控制红细胞执行反复的前进—后退的运动循环，即红细胞与细胞反复接触—分离的运动循环，并通过显示器的实时图像记录每次接触—分离过程中，细胞与红细胞的粘附状态。
膜片钳包括膜片钳放大器、膜片钳探头、膜片钳探头支架，膜片钳探头通过膜片钳探头支架固定于实验平台上，膜片钳探头的记录电极通过BNC转接线连接到玻璃电极夹持器末端，玻璃电极连接到玻璃电极夹持器的夹持端，玻璃电极夹持器安装在第二三维显微操作器上；参比电极通过实验容腔骨架侧面通孔接入到实验容腔内的细胞外液中。
荧光成像模块中，明场汞灯光源发射的全谱光通过明场入射光滤色片发出特定波段的明场光路，朝下正下方照射实验容腔，明场汞灯光源和实验容腔之间布置有明场入射光滤色片，实验平台安装在倒置显微镜的物镜上方，倒置显微镜的物镜正对实验容腔中心，倒置显微镜内部设有荧光光路入射光二色分光镜和全反射镜；倒置显微镜上部和下部侧面均开设有光路开口，倒置显微镜下部侧面光路开口连接分光器的一端，分光器内设有荧光光路出射光二色分光镜，分光器另一端的两个光路开口分别连接安装高速工业相机和荧光相机；明场汞灯光源朝下正下方发出光束经明场入射光滤色片后形成明场光路照射实验容腔中的细胞，透射后依次经倒置显微镜顶部的物镜、荧光光路入射光二色分光镜后经全反射镜反射到分光器内部的荧光光路出射光二色分光镜，再透射过荧光 光路出射光二色分光镜随后被高速工业相机接收；倒置显微镜上部侧面光路开口连接工业相机，明场光路在倒置显微镜内部进行分光，其中20％的光束经由光路开口入射到工业相机上，工业相机连接显示器；荧光光源发射的全谱光经倒置显微镜上部侧面接口入射到倒置显微镜内部的荧光入射光滤色片形成固定波段的荧光入射光，随后经过荧光光路入射光二色分光镜反射到实验容腔中的细胞，经细胞中的荧光蛋白/荧光染料发出的荧光发射光透过倒置显微镜的物镜返回到荧光光路入射光二色分光镜发生透射，再经全反射镜反射到分光器内部的荧光光路出射光二色分光镜，由荧光光路出射光二色分光镜反射后被荧光相机接收，形成荧光出射光路。
本发明通过上述装置能实现在不同膜电位控制下，细胞与红细胞粘附状态的记录，同时采集高信噪比的荧光图像来研究膜蛋白的跨膜信号传导。
还包括电脑主机，记录电极经玻璃电极夹持器、BNC转接线连接到膜片钳放大器上，膜片钳放大器通过USB接口连接到电脑主机，压电运动平台经压电运动平台控制器和电脑主机连接，荧光光源、荧光相机通过USB接口直接和电脑主机连接。
具体实施是以绿色荧光蛋白光谱特性检测为例，所述的明场汞灯光源发射全谱光；所述的明场入射光滤色片波段为617/73nm；所述的荧光入射光滤色片波段为465-495nm、荧光光路入射光二色分光镜的波段为505nm(波长小于505nm的光被反射，波长大于505n’m的光被透射)、荧光光路出射光二色分光镜的波段为580nm(波长小于580nm的光被反射，波长大于580nm的光被透射)。
所述的实验容腔骨架侧面预留开设了侧面通孔，膜片钳探头的参比电极穿设于侧面通孔布置，并稳定接入实验容腔内的细胞外液中。
本发明对荧光成像技术、膜片钳技术、微吸管进行了整合。整合后的实验控制程序包含了荧光成像技术、膜片钳技术、微吸管的必要功能，能够在同步记录三谱相信息的同时记录三谱相信息的耦合关系，可以离线还原微吸管技术所控制的碰撞状态、电压控制信号、电流采样信号、荧光谱采样图像在同一时间尺度上的对应关系。整合后的荧光—膜片钳—微吸管检测装置能够通过电脑主机对压电运动平台、膜片钳放大器、荧光光源、荧光相机等部件实施协同控制。
本发明具有的有益效果是：
本发明能够在检测细胞膜电位变化对膜蛋白相互作用的影响的同时实时观测其对膜蛋白跨膜信号传导的影响。本发明能够在同步记录荧光谱、电生理谱、粘附状态谱等三个谱相信息的同时记录三谱相信息间的耦合关系。
本发明主要针对生命科学领域中膜电位变化对膜蛋白动态功能的影响以及膜蛋白的跨膜信号传导。本发明具有以下优势：
1)能够直接检测获得膜电位变化对膜蛋白动态功能的影响；
2)能够根据荧光蛋白/荧光染料特性特异性地改变荧光光路中的滤色片等光学元件，得到高信噪比的荧光图像来研究膜蛋白的跨膜信号传导；
2)能够在同步记录三谱相信息的同时记录三谱相信息的耦合关系，可以用于离线还原微吸管所控制的碰撞状态、电压控制信号、电流采样信号、荧光谱采样图像在同一时间尺度上的对应关系。
附图说明
图1是本发明的系统设计图。
图2是本发明的实验原理图。
图3是本发明所涉及的实验容腔骨架设计图。
图中：1、明场汞灯光源，2、明场入射光滤色片，3、明场光路，4、荧光光路入射光二色分光镜，5、用于观测粘附状态的工业相机，6、用于观测粘附状态的显示器，7、全反射镜，8、分光器，9、荧光光路出射光二色分光镜，10、高速工业相机，11、荧光光源，12、荧光光源11发射的全谱光，13、荧光入射光滤色片，14、荧光入射光路，15、荧光出射光路，16、荧光相机，17、电脑主机，18、压电运动平台控制器，19、压电运动平台，20、膜片钳放大器，21、微吸管夹持器，22、实验容腔，23、控制微吸管的第一三维显微操作器，24、控制玻璃电极的第二三维显微操作器，25、玻璃电极，26、玻璃电极夹持器，27、BNC转接线，28、倒置显微镜，29、记录电极，30、电极内液，31实验平台，32、处于全细胞记录模式下的荧光细胞，33、红细胞，34、微吸管，35、容腔底面玻璃片，36、实验容腔骨架，37、侧面通孔。
具体实施方式
下面结合附图和实施例对本发明作进一步说明。
如图1，2所示，具体实施的装置包括压电运动平台19、微吸管夹持器21、实验容腔22、第一三维显微操作器23、第二三维显微操作器24、玻璃电极25、记录电极29、实验平台31、微吸管34和实验容腔骨架36；实验平台31上布置有实验容腔22、第一三维显微操作器23、第二三维显微操作器24、膜片钳探头支架等。如图3所示，实验容腔骨架36位于实验平台31中央，实验容腔骨架36表面粘贴有上下两片平行的玻璃片，两片玻璃片形成实验容腔22，实验容腔22的两侧镂空，供微吸管34和玻璃电极25进入；
本发明对实验容腔骨架36的结构进行了重新设计。具体地，为了更容易形成对细胞的高阻封接，本发明通过增大了实验容腔骨架36上下两玻璃片间的厚度来增加玻璃电极25可倾斜的角度。实验容腔骨架36侧面预留开设了侧面通孔37，膜片钳探头上的参比电极通过侧面通孔37接入到实验容腔内的细胞外液中。
实验容腔22的底面35上有处于全细胞记录模式下的荧光细胞32，微吸管34吸取红细胞33，细胞32和红细胞33处于实验容腔22内的细胞外液中，玻璃电极25内充有电极内液30，微吸管34连接微吸管夹持器21的夹持端，微吸管夹持器21安装在压电运动平台19上，压电运动平台19安装在第一三维显微操作器23上，第一三维显微操作器23固定于实验平台31上；压电运动平台19可以通过控制微吸管34带动红细胞33的运动，控制红细胞33执行反复的前进—后退的运动循环，即红细胞33与细胞32反复接触—分离的运动循环，并通过显示器6的实时图像记录每次接触—分离过程中，细胞32与红细胞33的粘附状态。
膜片钳相关部件包括膜片钳放大器20、膜片钳探头、膜片钳探头支架，膜片钳探头通过膜片钳探头支架固定于实验平台31上，膜片钳探头的记录电极通过BNC转接线27连接到玻璃电极夹持器26末端，玻璃电极25连接到玻璃电极夹持器26的夹持端，玻璃电极夹持器26安装在第二三维显微操作器24上，第二三维显微操作器24固定在实验平台31上；参比电极通过实验容腔骨架36侧面通孔37接入到实验容腔内的细胞外液中。玻璃电极25伸入到实验容腔内的电极外液中，记录电极29末端插入到玻璃电极25内灌注的电极内液30中
第一三维显微操作器23和第二三维显微操作器24均为可以通过手柄操作的超精密电动三维运动平台。
荧光成像模块中，主要包括明场汞灯光源1、明场入射光滤色片2、明场光路3、荧光光路入射光二色分光镜4、用于观测粘附状态的工业相机5、用于观测粘附状态的显示器6、全反射镜7、分光器8、荧光光路出射光二色分光镜9、高速工业相机10、荧光光源11、荧光光源11发射的全谱光12，荧光入射光滤色片13，荧光入射光路14，荧光出射光路15、荧光相机16和倒置显微镜28。
如图1所示，本发明在荧光成像上具有荧光光源11、荧光相机16等设备，并根据荧光蛋白/荧光染料的激发、发射光谱在倒置显微镜的光路增加了滤光片、反光镜、二色分光镜、分光器等光学设备，将荧光光路的波段与明场光波段分开来增强荧光图像的信噪比。
具体地，明场汞灯光源1发射的全谱光通过明场入射光滤色片2发出特定 波段的明场光路3，朝下正下方照射实验容腔22，明场汞灯光源1和实验容腔22之间布置有明场入射光滤色片2，实验平台31安装在倒置显微镜28的物镜上方，倒置显微镜28的物镜正对实验容腔22中心，倒置显微镜28内部设有荧光光路入射光二色分光镜4和全反射镜7；倒置显微镜28上部和下部侧面均设有光路接口，倒置显微镜28下部侧面接口连接分光器8的一端，分光器8内设有荧光光路出射光二色分光镜9，分光器8另一端的两个接口分别连接安装高速工业相机10和荧光相机16；明场汞灯光源1朝下正下方发出光束经明场入射光滤色片2后形成明场光路3照射实验容腔22中的细胞，透射后依次经倒置显微镜28顶部的物镜、荧光光路入射光二色分光镜4后经全反射镜7反射到分光器8内部的荧光光路出射光二色分光镜9，再透射过荧光光路出射光二色分光镜9随后被高速工业相机10接收；倒置显微镜28上部侧面接口连接工业相机5，明场光路3在倒置显微镜28内部进行分光，其中20％的光束经由该接口入射到工业相机5上，工业相机5连接显示器6；荧光光源11发射的全谱光12经倒置显微镜28上部侧面接口入射到倒置显微镜28内部的荧光入射光滤色片13形成指定波段的荧光入射光14，随后经过荧光光路入射光二色分光镜4反射到实验容腔22中；细胞中的荧光蛋白/荧光染料发出的荧光发射光透过倒置显微镜28的物镜回到荧光光路入射光二色分光镜4发生透射，再经全反射镜7反射到分光器8内部的荧光光路出射光二色分光镜9，并由荧光光路出射光二色分光镜9反射后被荧光相机16接收，形成荧光出射光路15；
本发明通过上述装置能实现在不同膜电位控制下，细胞与红细胞粘附状态的记录，同时采集高信噪比的荧光图像来研究膜蛋白的跨膜信号传导。
本发明还包括电脑主机17，记录电极29经玻璃电极夹持器26、BNC转接线27连接到膜片钳放大器20上，膜片钳放大器20通过USB接口连接到电脑主机17，压电运动平台19经压电运动平台控制器18和电脑主机17连接，荧光光源11、荧光相机16通过USB接口直接和电脑主机17连接。通过电脑主机17对压电运动平台控制器18、膜片钳放大器20、荧光光源11、荧光相机16等部件实施协同控制。
以绿色荧光蛋白光谱特性为例，本发明：
所述的明场汞灯光源1发射全谱光。
所述的明场入射光滤色片2波段为617/73nm。
所述的荧光入射光滤色片13波段为465-495nm、荧光光路入射光二色分光镜4的波段为505nm，波长小于505nm的光被反射，波长大于505nm的光被透 射；荧光光路出射光二色分光镜9的波段为580nm，波长小于580nm的光被反射，波长大于580nm的光被透射。
本发明所述的实验容腔骨架36侧面预留开设了侧面通孔37，膜片钳探头的参比电极通过侧面通孔37稳定接入实验容腔22内的细胞外液中。
本发明的具体实施工作过程如下：
本发明基于荧光—膜片钳—微吸管检测装置的实验目的设计了新的实验方案。
具体地，实验容腔22的准备过程中，表达目标膜蛋白分子及胞内荧光蛋白的的悬浮细胞32通过多聚赖氨酸粘附在实验容腔底面的玻璃片35上，处于半贴壁状态；
表面连接另一蛋白质分子的红细胞33加入到实验容腔22内的细胞外液中；
实验操作人员选取表面光滑、荧光信号较强的目标细胞32，并通过移动倒置显微镜28的载物台将其移动到视野中间，随后通过操控第二三维显微操作器24控制玻璃电极25对目标细胞32执行封接、破膜、补偿等常规全细胞记录膜片钳的操作；
实验操作人员通过操控第一三维显微操作器23控制微吸管34吸取一个红细胞33；
实验操作人员设置电压刺激参数(波形、幅值、频率等)、微吸管实验参数(红细胞33与细胞32的接触时间等)、荧光成像模块采样参数(曝光时间、采样间隔等)；
开始数据采集阶段：程序自动对全细胞记路模式下的细胞32施加设定的电压刺激，程序自动显示、记录当前电压、电流信息；程序协同控制荧光光源11、荧光相机16，按照设定的荧光参数(曝光时间、采样间隔等)进行自动采样，每次采样结束后，荧光谱的图像以强度数组的形式在主机17的硬盘中自动存储为独立文件；程序控制红细胞33执行与细胞32反复接触—分离的运动循环，实验操作人员通过显示器6的实时图像记录每次接触—分离过程中，细胞32与红细胞33的粘附状态。
记录完成后(记录时长为50次接触—分离的运动循环的时间)保存当次记录过程中电压、电流数据，以及手动记录的粘附状态(其粘附状态出现的频率记为当前膜电位刺激条件下的粘附频率)。
实验操作人员随后根据实验设计修改电压刺激参数，重复数据采集阶段；
通过对比不同电压刺激下粘附频率的变化，以及细胞中荧光信号随着时间的变化，这种整合膜片钳的微吸管技术可以解析膜电位变化对膜蛋白相互作用的影响及其对膜蛋白跨膜信号传导的影响。
特别地，本发明能够在同步记录荧光谱、电生理谱、粘附状态谱等三谱相信息的同时记录三谱相信息的耦合关系。具体地，程序开始数据采集阶段的同时，荧光相机采集第一张图像(此时粘附状态谱控制红细胞33开始运动，电生理谱开始输出电压控制信号)，随后按照固定的时间间隔进行采样；此后压电运动平台19每次运动状态变化(开始运动或停止运动)的时刻，电生理谱的数据索引都会被记入一个独立的索引数组文件中，该索引数组文件可以用于离线还原微吸管技术所控制的红细胞33的运动状态与电压控制信号、电流采样信号在同一时间尺度上的对应关系。这样一来，三个谱相的信息可以再同一时间尺度上的对应，三谱相信息的耦合关系也能被解析。
上述具体实施方式用来解释说明本发明，而不是对本发明进行限制，在本发明的精神和权利要求的保护范围内，对本发明作出的任何修改和改变，都落入本发明的保护范围。

Claims (4)

  1. 一种荧光—膜片钳—微吸管检测装置，其特征在于：包括实验平台(31)、明场汞灯光源(1)、倒置显微镜(28)、玻璃电极(25)、微吸管(34)、压电运动模块(19)、第一三维显微操作器(23)、记录电极(29)和实验容腔骨架(36)；实验平台(31)上布置有实验容腔(22)、第一三维显微操作器(23)、第二三维显微操作器(24)和膜片钳，实验容腔骨架(36)位于实验平台(31)中央，实验容腔骨架(36)放置有上下两片平行的玻璃片，两片玻璃片中间形成实验容腔(22)，实验容腔(22)的两侧镂空，供微吸管(34)和玻璃电极(25)进入；实验容腔(22)的底面(35)上有处于全细胞记录模式下的荧光细胞(32)，微吸管(34)吸取红细胞(33)，细胞(32)和红细胞(33)处于实验容腔(22)内的细胞外液中，玻璃电极(25)内充有电极内液(30)，微吸管(34)连接微吸管夹持器(21)的夹持端，微吸管夹持器(21)安装在压电运动平台(19)上，压电运动平台(19)安装在第一三维显微操作器(23)上，第一三维显微操作器(23)固定于实验平台(31)上；膜片钳包括膜片钳放大器(20)、膜片钳探头、膜片钳探头支架，膜片钳探头通过膜片钳探头支架固定于实验平台(31)上，膜片钳探头的记录电极通过BNC转接线(27)连接到玻璃电极夹持器(26)末端，玻璃电极(25)连接到玻璃电极夹持器(26)的夹持端，玻璃电极夹持器(26)安装在第二三维显微操作器(24)上；参比电极接入到实验容腔内的细胞外液中；
    荧光成像模块中，明场汞灯光源(1)发射的全谱光通过明场入射光滤色片(2)发出特定波段的明场光路(3)，朝下正下方照射实验容腔(22)，明场汞灯光源(1)和实验容腔(22)之间布置有明场入射光滤色片(2)，实验平台(31)安装在倒置显微镜(28)的物镜上方，倒置显微镜(28)的物镜正对实验容腔(22)中心，倒置显微镜(28)内部设有荧光光路入射光二色分光镜(4)和全反射镜(7)；倒置显微镜(28)上部和下部侧面均开设有光路开口，倒置显微镜(28)下部侧面光路开口连接分光器(8)的一端，分光器(8)内设有荧光光路出射光二色分光镜(9)，分光器(8)另一端的两个光路开口分别连接安装高速工业相机(10)和荧光相机(16)；明场汞灯光源(1)朝下正下方发出光束经明场入射光滤色片(2)后形成明场光路(3)照射实验容腔(22)中的细胞，透射后依次经倒置显微镜(28)顶部的物镜、荧光光路入射光二色分光镜(4)后经全反射镜(7)反射到分光器(8)内部的荧光光路出射光二色分光镜(9)，再透射过荧光光路出射光二色分光镜(9)随后被高速工业相机 (10)接收；倒置显微镜(28)上部侧面光路开口连接工业相机(5)，明场光路(3)在倒置显微镜(28)内部进行分光，光束经由光路开口入射到工业相机(5)上，工业相机(5)连接显示器(6)；荧光光源(11)发射的全谱光(12)经倒置显微镜(28)上部侧面接口入射到倒置显微镜(28)内部的荧光入射光滤色片(13)形成固定波段的荧光入射光(14)，随后经过荧光光路入射光二色分光镜(4)反射到实验容腔(22)中的细胞，经细胞中的荧光蛋白/荧光染料发出的荧光发射光透过倒置显微镜(28)的物镜返回到荧光光路入射光二色分光镜(4)发生透射，再经全反射镜(7)反射到分光器(8)内部的荧光光路出射光二色分光镜(9)，由荧光光路出射光二色分光镜(9)反射后被荧光相机(16)接收，形成荧光出射光路(15)。
  2. 根据权利要求1所述的一种荧光—膜片钳—微吸管检测装置，其特征在于：还包括电脑主机(17)，记录电极(29)经玻璃电极夹持器(26)、BNC转接线(27)连接到膜片钳放大器(20)上，膜片钳放大器(20)连接到电脑主机(17)，压电运动平台(19)经压电运动平台控制器(18)和电脑主机(17)连接，荧光光源(11)、荧光相机(16)直接和电脑主机(17)连接。
  3. 根据权利要求1所述的一种荧光—膜片钳—微吸管检测装置，其特征在于：具体实施是以绿色荧光蛋白光谱特性检测为例，所述的明场汞灯光源(1)发射全谱光；所述的明场入射光滤色片(2)波段为617/73nm；所述的荧光入射光滤色片(13)波段为465-495nm、荧光光路入射光二色分光镜(4)的波段为505nm、荧光光路出射光二色分光镜(9)的波段为580nm。
  4. 根据权利要求1所述的一种荧光—膜片钳—微吸管检测装置，其特征在于：所述的实验容腔骨架(36)侧面预留开设了侧面通孔(37)，膜片钳探头的参比电极穿设于侧面通孔(37)布置，并稳定接入实验容腔(22)内的细胞外液中。
PCT/CN2020/115249 2019-12-11 2020-09-15 一种荧光—膜片钳—微吸管检测装置 WO2021114790A1 (zh)

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CN111122525B (zh) * 2019-12-11 2021-01-15 浙江大学 一种荧光—膜片钳—微吸管检测装置
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1458972A (zh) * 2001-01-09 2003-11-26 松下电器产业株式会社 测量细胞外电势的装置、利用该装置测量细胞外电势的方法以及配有该装置的快速筛选药物的仪器
US20070264634A1 (en) * 2002-10-10 2007-11-15 Nanosys, Inc. Nano-chem-fet based biosensors
CN102119331A (zh) * 2008-06-05 2011-07-06 生命科技公司 细胞跨膜电位的激活和监测
CN104024839A (zh) * 2011-12-20 2014-09-03 独立行政法人科学技术振兴机构 平面膜片钳装置、该装置用电极部及细胞离子通道电流测量方法
CN109929900A (zh) * 2019-03-01 2019-06-25 天津工业大学 Lf_emf诱发细胞膜电位调控离子通道电活动的方法
CN111122525A (zh) * 2019-12-11 2020-05-08 浙江大学 一种荧光—膜片钳—微吸管检测装置

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2975900A (en) * 1999-01-27 2000-08-18 Regents Of The University Of California, The Assays for sensory modulators using a sensory cell specific g-protein alpha subunit
US7444856B2 (en) * 2004-09-23 2008-11-04 The Board Of Trustees Of The Leland Stanford Junior University Sensors for electrochemical, electrical or topographical analysis
CN100445397C (zh) * 2006-12-14 2008-12-24 上海交通大学 电磁力控制单链核酸穿孔速度的方法与装置
JP5307353B2 (ja) * 2007-04-26 2013-10-02 オリンパス株式会社 多光子励起レーザ走査型顕微鏡および多光子励起蛍光画像取得方法
CN201569654U (zh) * 2009-11-19 2010-09-01 浙江大学 检测细胞生理参数的光电复合一体式传感器及集成硅芯片
CN206512221U (zh) * 2016-12-20 2017-09-22 浙江大学 一种细胞囊泡快速标记装置
US11073508B2 (en) * 2017-03-28 2021-07-27 Arizona Board Of Regents On Behalf Of The University Of Arizona Rapid conductance based ion channel analysis
CN107314978B (zh) * 2017-07-28 2023-05-09 浙江大学 微区可见光谱仪及光谱测量方法
CN108913599B (zh) * 2018-08-10 2021-08-17 清华大学 一种活细胞原位培养长时程多模信息检测方法及系统

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1458972A (zh) * 2001-01-09 2003-11-26 松下电器产业株式会社 测量细胞外电势的装置、利用该装置测量细胞外电势的方法以及配有该装置的快速筛选药物的仪器
US20070264634A1 (en) * 2002-10-10 2007-11-15 Nanosys, Inc. Nano-chem-fet based biosensors
CN102119331A (zh) * 2008-06-05 2011-07-06 生命科技公司 细胞跨膜电位的激活和监测
CN104024839A (zh) * 2011-12-20 2014-09-03 独立行政法人科学技术振兴机构 平面膜片钳装置、该装置用电极部及细胞离子通道电流测量方法
CN109929900A (zh) * 2019-03-01 2019-06-25 天津工业大学 Lf_emf诱发细胞膜电位调控离子通道电活动的方法
CN111122525A (zh) * 2019-12-11 2020-05-08 浙江大学 一种荧光—膜片钳—微吸管检测装置

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