Fluorescence microscope capable of being used for detecting in-situ single-molecule fluorescence-current signal and detection method

Abstract

The invention provides a fluorescence microscope and a detection method which can be used for detecting in-situ single-molecule fluorescence-current signals. On the basis of a single-channel recording patch clamp technology, fluorescence microscope equipment and related fluorescence technology support are matched, so that an electric signal and a fluorescence signal of a clamping area, namely a single-channel recording area, of the patch clamp are observed at the same time. According to the invention, functional information and structural information of ion channels and other membrane proteins can be simultaneously analyzed in real time and in situ by combining a single-channel recording patch clamp technology.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological macromolecular dynamics information detection or ion channel electrophysiological characteristics detection, and in particular relates to a fluorescence microscope and a detection method that can be used to detect in situ single-molecule fluorescence-current signals. Background Art

[0002] Electrical signals are a crucial component of life and one of the most crucial physiological characteristics underlying human intelligence. The most fundamental components of electrical signals in life are membrane proteins called ion channels and ion pumps, and understanding how they work has long been a topic of great interest.

[0003] Among existing technologies, the technology that can analyze the electrical properties of ion channels and ion pumps is relatively mature. After years of development, the single-channel recording patch clamp technology can record the process of ion channels (pumps) functioning at an extremely high sampling rate. It can clearly know the voltage on both sides of the membrane and the current passing through the membrane when a channel functions.

[0004] Technologies capable of analyzing membrane protein structures have also made significant progress in recent years. The advent of cryo-electron microscopy has revolutionized the resolution of biomolecular structures, enabling sub-nanometer resolution. However, due to limitations in its principles, it cannot provide information on the dynamics of biomolecules.

[0005] Fluorescence microscopy is a relatively effective tool for analyzing biomolecular dynamics. Fluorescence resonance energy transfer (FRET), along with its derivatives SIFA, Lipo-FRET, and Queen-FRET, all offer methods for observing biomolecular conformation and positional changes at the single-molecule level. Their spatial resolution can reach subnanometers, and their temporal resolution can reach submilliseconds. Fluorescence microscopy plays an irreplaceable role in understanding the structural changes of biomolecules.

[0006] Although the above technologies help us analyze the structural and functional information of ion channels (pumps), people are still unable to simultaneously observe the connection between the functional information (electrical information) and structural information (kinetic information) when ion channels function. The two are always unable to match, which leads to a blind spot in the understanding of ion channels (pumps). Summary of the Invention

[0007] Therefore, the purpose of the present invention is to overcome the defects of the above-mentioned prior art and provide a fluorescence microscope and detection method that can be used to detect in situ single-molecule fluorescence-current signals, so as to simultaneously analyze the electrical information and kinetic information of ion channels (pumps and other membrane proteins), thereby revealing the last blind spot for further understanding of ion channels (pumps).

[0008] The present invention provides a fluorescence microscope, comprising:

[0009] a laser that emits a parallel light beam in a specified direction;

[0010] a beam shaping component, the beam shaping component being arranged on a laser emission path of the laser to form the parallel light beam into a parallel annular light beam;

[0011] a beam guiding assembly disposed on a laser emission path of the laser; and

[0012] objective lens;

[0013] The beam guiding assembly guides the annular beam to the objective lens and focuses it on the imaging focal plane of the objective lens to form a focal spot. The focal spot is focused on the sample to be observed to generate a light signal, and the light signal is imaged through the objective lens and the beam guiding assembly.

[0014] According to the fluorescence microscope of the present invention, preferably, the axis of the beam shaping component coincides with the axis of the laser.

[0015] According to the fluorescence microscope of the present invention, preferably, the light beam guiding component is a dichroic mirror.

[0016] According to the fluorescence microscope of the present invention, preferably, the laser is a continuous laser or a pulsed laser.

[0017] According to the fluorescence microscope of the present invention, preferably, the beam forming assembly includes: a convex lens, an aperture and a concave lens; the axes of the convex lens, the aperture and the concave lens coincide with the axis of the beam forming assembly, and are arranged in sequence along the light incident direction of the beam forming assembly.

[0018] According to the fluorescence microscope of the present invention, the front focal planes of the convex lens and the concave lens are aligned.

[0019] The present invention is based on the single-channel recording patch clamp technology and is supported by the fluorescence microscope device to achieve simultaneous observation of the clamping area of ​​the patch clamp, that is, the electrical signal and the fluorescence signal of the single-channel recording area.

[0020] The single-channel recording patch clamp technique attaches a prefabricated pipette to the surface of the cell membrane and forms a tight seal (resistance reaches GΩ level) with the cell membrane. The area where the pipette and the cell membrane are sealed is the clamping area. The electrodes in the cell bath and the electrodes in the pipette are used to record the potential on both sides of the clamping area and the current passing through this area. For suitable cells, it can be ensured that there is only one ion channel (pump) in the clamping area, thereby realizing the recording of electrical signals of a single channel, which is single-channel recording.

[0021] In existing fluorescence imaging technologies, fluorescence signals can reflect sub-nanostructure changes of biomolecules through FRET and its derivative technologies.

[0022] The energy transfer efficiency E between the donor and acceptor in FRET is defined as:

[0023]

[0024]

[0025]

[0026] τ D =1 / k D (4)

[0027]

[0028] Where R is the distance between the two fluorescent probes, R0 is the characteristic quenching distance, and the distance corresponding to the transfer efficiency E of 50%. Since the distance between the donor and the acceptor R is inversely proportional to the sixth power, E is very sensitive to the spatial distance, which is why fluorescence resonance transfer can be used to measure nanometer-scale distances. R0 is calculated by the dipole approximation, where Q D is the donor quantum yield, n is the refractive index of the medium, κ 2 is the dipole orientation factor between the two fluorescent molecules, ε A is the maximum extinction coefficient of the acceptor, J(λ) is the normalized donor emission spectrum F D and the receptor absorption spectrum E A The spectral overlap coefficient, k D represents the fluorescence emission rate of the donor molecule.

[0029] Based on the above techniques and principles, the present invention provides a method for combined detection of in situ single-molecule fluorescence and current signals using the above-mentioned fluorescence microscope, the steps of which are as follows:

[0030] Step 1: Select appropriate fluorescence imaging technology based on the detection requirements, and accordingly select the type of dye used for fluorescent labeling and the number of labels; use the selected dye to label the channel protein to be observed;

[0031] Step 2: assembling the channel protein onto the cell membrane surface and ensuring that it can perform its normal function and the normal physiological process of the cell will not be significantly disturbed by the channel protein; the expression density of the channel protein is in the range of 0.1-10 μm2;

[0032] Step 3: Selecting a suitable activation signal to activate the channel protein; a channel protein with a basal activity of 0 does not need to be activated; forming a single-channel recording area on the cell membrane surface and recording its electrical signal according to the single-channel recording patch clamp technical specifications; aligning the single-channel recording area with the fluorescence imaging focal spot of the fluorescence microscope, performing single-molecule fluorescence signal imaging on the single-channel recording area with the fluorescence microscope, and simultaneously recording its fluorescence signal;

[0033] Step 4: Analyze the fluorescence signal to extract the kinetic information of the channel protein; the electrical signal reflects the functional information of the channel protein; align the two in time to reflect the relationship between the kinetic information and the functional information of the channel protein.

[0034] According to the in situ single-molecule fluorescence-current signal detection method of the present invention, preferably, in step 1: quantum dots or gold nanoparticles are used as markers instead of fluorescent markers.

[0035] According to the in situ single-molecule fluorescence-current signal detection method of the present invention, preferably, in step 2: the channel protein is assembled onto the cell membrane surface by transfection, and it is ensured that it can perform its normal function.

[0036] According to the in situ single-molecule fluorescence-current signal detection method of the present invention, preferably, in step 3: a trigger signal is set so that the fluorescence signal record is accompanied by trigger information to ensure that the two measurement signals can be aligned.

[0037] According to the in situ single-molecule fluorescence-current signal detection method of the present invention, preferably, step 3 includes:

[0038] Step 301, adjusting the fluorescence microscope to align the region of the channel protein to be observed with the focal spot;

[0039] Step 302 , according to the single-channel recording patch clamp technical specifications, move the pipette tip to the area to be observed;

[0040] Step 303, applying a test voltage according to the single-channel patch clamp technical specifications, lowering the pipette to form a single-channel seal at its tip, with an impedance greater than 10 GΩ, and forming the single-channel recording area;

[0041] Step 304, start single-channel recording and remove the test voltage;

[0042] Step 305 , adjusting the fluorescence microscope, selecting weak laser excitation, and adjusting the objective lens so that the signal to be observed at the tip of the pipette is clear;

[0043] Step 306, adjusting the fluorescence microscope, selecting strong laser excitation, applying a channel protein activation signal, and completing the recording of the fluorescence signal and the electrical signal;

[0044] The single-channel recording area formed in step 303 may not include the target single channel. If this happens, the process should return to step 301.

[0045] According to the in situ single-molecule fluorescence-current signal detection method of the present invention, preferably, in step 303, for nerve cells that have a membrane potential themselves, or channels with a certain potential in a resting state, a corresponding potential needs to be applied for sealing; if it is an ion channel that is sensitive to mechanical force, the positive pressure of the seal needs to be slowly removed to give the cell membrane time to relax the tension, and negative pressure cannot be applied arbitrarily.

[0046] According to the in-situ single-molecule fluorescence-current signal detection method of the present invention, preferably, the fluorescence information recording instrument in step 306 is an EMCCD or a single-photon detector.

[0047] According to the in situ single-molecule fluorescence-current signal detection method of the present invention, preferably, the channel is a voltage-gated ion channel, and a suitable voltage signal is selected to drive it; or the channel is a mechanical force-sensitive ion channel, and the application of mechanical force is controlled to cooperate with activation.

[0048] Compared with the prior art, the advantages of the present invention are:

[0049] 1. The present invention is based on the single-channel recording patch clamp technology, and is supported by the fluorescence microscope device to achieve simultaneous observation of the electrical signal and fluorescence signal in the clamped area of ​​the patch clamp, that is, the single-channel recording area, so that the working process of the ion channel can be fully analyzed;

[0050] 2. The present invention is compatible with a variety of fluorescence imaging technologies in terms of fluorescence imaging, so as to achieve a multifaceted description of membrane protein dynamics information;

[0051] 3. The technical principle of the present invention limits the free diffusion of ion channels, which enables some technologies that improve temporal resolution to be further implemented on top of this technology. Here is a simple example: the EMCCD used to collect fluorescence signals can use a smaller exposure frame to shorten the recording time required for a single frame. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The embodiments of the present invention are further described below with reference to the accompanying drawings, in which:

[0053] Figure 1 is a schematic structural diagram of a fluorescence microscope 100 according to an embodiment of the present invention;

[0054] Figure 2 is a structural diagram of a beam shaping assembly 102 according to an embodiment of the present invention;

[0055] Figure 3 Schematic diagram of DNA 100 pM fluorescence-current signal detection according to an embodiment of the present invention;

[0056] Figure 4 Schematic diagram of mPiezo1 fluorescence-current signal detection according to an embodiment of the present invention. DETAILED DESCRIPTION

[0057] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below through specific embodiments in conjunction with the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0058] Figure 1 FIG. 1 is a schematic structural diagram of a fluorescence microscope 100 that can be used for in-situ single-molecule fluorescence-current signal detection.

[0059] like Figure 1 As shown, fluorescence microscope 100 generally includes a laser 101, a beam shaping assembly 102, a beam steering assembly 103, and an objective lens 104. Laser 101 emits a parallel light beam in a specified direction. Beam shaping assembly 102 is disposed in the laser emission path of laser 101. The parallel light beam is formed into a parallel annular light beam 105 by beam shaping assembly 102. Beam steering assembly 103 is disposed in the laser emission path of laser 101 and forms a certain angle with parallel annular light beam 105. Objective lens 104 is disposed in the output light path of beam steering assembly 103. Beam steering assembly 103 is configured to guide annular light beam 105 to objective lens 104, and focus it on the imaging focal plane of objective lens 104 to form a focal spot 201. Focal spot 201 is focused on the observed area 301 of sample 501 to be observed, generating an optical signal. The optical signal is transmitted through objective lens 104 and beam steering assembly 103 to form an image.

[0060] During testing, the fluorescence microscope 100 is turned on, and the sample 501 to be tested is placed under the objective lens 104. The weak laser light of the laser 101 is turned on to confirm the focal plane of the objective lens 104. After the focal plane of the objective lens 104 is confirmed, the strong laser light of the laser 101 is turned on. The strong laser light is a parallel beam along a specified direction. The parallel beam passes through the beam shaping component 102 to form a parallel annular beam 105. The annular beam 105 is hollow, with no beam at the center. The size of the hollow portion or the circular portion of the annular beam 105 can be adjusted according to the test requirements.

[0061] An annular beam 105 is guided by the beam guiding assembly 103 to the objective lens 104, where it is focused on the imaging focal plane of the objective lens 104 to form a focal spot 201. Because the annular beam 105 is hollow, there is no beam at its center. Therefore, until the annular beam 105 is focused at a certain angle to form the focal spot 201, there is no beam at its center. The optical signal generated by the focal spot 201 focused on the observed area 301 of the sample 501 passing through the center of the annular beam 105 is not interfered with by other light. This improves the signal-to-noise level of the transmission imaging produced by the objective lens 104 and the beam guiding assembly 103.

[0062] In one or more embodiments, the beam shaping assembly 102 is coincident with the axis of the laser 101 .

[0063] In one or more embodiments, Figure 2 As shown, the beam shaping assembly 102 includes a convex lens 1021, an aperture 1022, and a concave lens 1023. The axes of the convex lens 1021, the aperture 1022, and the concave lens 1023 coincide with the axis of the beam shaping assembly 102, and are sequentially arranged along the incident direction of the light of the beam shaping assembly 102.

[0064] In one or more embodiments, the front focal planes of the convex lens 1021 and the concave lens 1023 are aligned.

[0065] In one or more embodiments, the beam guiding assembly 103 is a dichroic mirror. When light of different wavelengths reaches the dichroic mirror, it is reflected or transmitted with different reflectivities and transmittances. This enables selective reflection and transmission of light of different wavelengths, thereby achieving light beam separation and reorganization, and avoiding signal crosstalk.

[0066] In one or more embodiments, the laser 101 is a continuous laser or a pulsed laser.

[0067] like Figure 3As shown, this example uses the in situ single-molecule fluorescence-current signal detection method to demonstrate the FRET phenomenon and photobleaching process on DNA in a single-channel recording area. In this example, the cell membrane does not contain ion channels. The electrical signal primarily demonstrates the noise level in the absence of ion channels, while the fluorescence signal primarily demonstrates the FRET phenomenon and the photobleaching process of the fluorescent molecules, thereby verifying the signal-to-noise ratio of the fluorescence signal in this detection method.

[0068] 100pM of DNA with cholesterol and a dual fluorescent label at one end is incubated with cells, allowing the DNA to actively bind to the cell membrane. Specifically, this DNA has cholesterol at one end, the cholesterol end is hydrophobic, and the DNA end is hydrophilic. This structure will actively bind to the cell membrane structure when it encounters the cell membrane structure, with the cholesterol inserted into the cell membrane and the DNA end suspended in the aqueous solution. In addition, the DNA is labeled with two fluorescent groups, Cy3 and Cy5, and the distance between the two is designed to allow FRET to occur, with a stable FRET value (0.5). When a single-channel recording area is formed using the single-channel recording patch clamp technique, just like there is exactly one ion channel in the area, there may also be exactly one added DNA molecule. The single-channel recording area only has a fluorescent signal, and the electrical signal is a stable "0". In the embodiment, it can be seen that there is a two-color fluorescent signal in the single-channel recording area, there is a FRET effect between the two, and the measured FRET value is consistent with the design (here, it reflects the structure of the DNA), and the fluorescence signal is eventually bleached; in addition, this fluorescent signal is different from the free fluorescent signal and will be confined to an area of ​​approximately 7×7 pixels. The specific steps are as follows:

[0069] Step 1: Select FRET fluorescence imaging technology, and accordingly select dye Cy3 as the donor and dye Cy5 as the acceptor, and apply these two fluorescent labels to 100 pM cholesterol DNA molecules to form a FRET pair.

[0070] Step 2: Add 100 pM of the cholesterol DNA prepared in Step 1 to the cell culture medium at an appropriate concentration (typically 100 nM), ensuring that it can function normally and that the channel protein does not significantly interfere with the normal physiological processes of the cells. Incubate the cholesterol DNA at 100 pM for approximately 1 minute.

[0071] Step 3, prepare for single-channel patch clamp sealing, fill the prefabricated single-channel recording pipette with electrode liquid, and load it on the electrode holder. Apply a positive pressure of about 5 cm of water column to the pipette to ensure the patency of the pipette tip. Under the fluorescence microscope 100, select the cell and move it to the center of the field of view, slowly lower the pipette, and confirm the position of the pipette tip in the field of view. Turn on the laser briefly, move the cell so that the area to be observed 301 is aligned with the focal spot 201, and then move the pipette so that its tip is above the area to be observed 301. Then use the patch clamp 601 to start sealing, apply the test voltage, and when the pipette descends and the tip approaches the cell, slowly reduce the pipette pressure to 0 (sometimes some negative pressure can be applied), and the tip will quickly form a seal with an impedance greater than 10GΩ.

[0072] Start single-channel electrical and fluorescence signal recording: Remove the test voltage and begin recording the electrical signal. Simultaneously, perform dual-channel Cy3 / Cy5 imaging and record the fluorescence signal using an EMCCD. Before recording, confirm the focal plane with a weak laser, then begin the experiment at an appropriate intensity and record the sample's fluorescence signal.

[0073] It should be noted that the presence or absence of target fluorescent molecules in the formed sealing area is a random event. Therefore, when there is no available signal, it is necessary to return to step 2 and reselect the area for sealing.

[0074] Step 4, analyze the fluorescence signal: calculate the FRET value of the two molecules based on the recorded fluorescence signal, and calculate the distance between the two fluorescent molecules with the help of the fluorescence spectra of Cy3 and Cy5. In addition, if the recording time is longer, the target molecule can be seen to be bleached. At the same time, since the target molecule, that is, the fluorescently labeled cholesterol-DNA, does not have the function of forming a transmold pathway, the electrical signal will remain unchanged. The kinetic information extracted from the fluorescence signal and the functional information extracted from the electrical signal are aligned in time. Recording and analyzing the motion information of this single-molecule signal can reveal that its diffusion range is extremely limited, limited to a range of about 7×7 pixels, and its actual size just corresponds to the size of the prefabricated pipette tip (diameter ~1.2μm).

[0075] like Figure 4 As shown, this example uses the in situ single-molecule fluorescence-current signal detection method to measure the electrical and fluorescence signals of mPiezo1 in a single-channel recording region. In this example, HEK293 cells were transiently transfected with an mPiezo1 plasmid to express an appropriate amount of fluorescently labeled mPiezo1 channel protein on the HEK293 cell surface. Using single-channel patch clamp recording, the current signal of a single mPiezo1 channel and the bleaching process of its fluorescence signal were recorded.

[0076] Among them, mPiezo1 is a mechanically sensitive ion channel protein that responds to the tension of the cell membrane surface by opening itself, mediating the influx of calcium ions, and inactivating after a period of time, thereby regulating the life activities of the cell. In this embodiment, the cell membrane surface tension will be regulated by the pressure of the single-channel recording area. When the negative pressure of the single-channel recording area is increased, the probability of mPiezo1 opening in this area will increase, which will be recorded by the patch clamp. In terms of fluorescence signal, a signal quenching will be seen to indicate that there is only one ion channel in this area. The specific steps are as follows:

[0077] Step 1: Select a monochrome fluorescence imaging technique, and accordingly select a fluorescent protein as a fluorescent marker, and label the mPiezo1 channel protein with the fluorescent protein.

[0078] Step 2: Transiently transfect HEK293 cells containing the mPiezo1 gene and incubate them to express the channel protein. This protein is fluorescent, so cells that have been successfully transfected and expressing the protein can be confirmed under a fluorescence microscope at 100° for subsequent experiments.

[0079] Step 3: Prepare for single-channel patch clamp sealing. The steps are the same as Step 3 in Example 1. In this example, a mechanosensitive ion channel is used, so careful pressure control is crucial. The transition from positive pressure to negative pressure should be slow, allowing the cell membrane time to relax and release tension. After the seal is established, the pipette pressure should be returned to zero and maintained for a period of time.

[0080] It should be noted that the process of forming single-channel recording seals for different ion channels has different contents that need to be paid attention to, and step 3 in Example 1 is the most basic process.

[0081] Start single-channel electrical and fluorescence signal recording: Turn on the weak laser light of the fluorescence microscope 100, adjust the objective lens 104, align the focal spot 201, and then switch to the strong laser light. Apply a force signal to activate the ion channel; specifically, apply a pressure of approximately -1000 Pa. Remove the test voltage and begin recording the electrical signal and fluorescence signal simultaneously. For this example, only the single fluorescence channel recording is sufficient.

[0082] Step 4. The electrical signal will change with the sudden change of negative pressure in the pipette, and the patch clamp system will record the electrical signal of a single channel to characterize the state of channel opening (opening time, current size, dwell time, etc.). The fluorescence signal here is mainly used as a reference for signal-to-noise ratio measurement and does not contain kinetic information. The bleaching process can be seen in long-term recordings. The kinetic information extracted from the fluorescence signal and the functional information extracted from the electrical signal are aligned in time. In addition, it can also be found in the recording and analysis of the motion information of this single-molecule signal that its diffusion range is extremely limited, limited to a range of about 7×7 pixels, and its actual size just corresponds to the size of the prefabricated pipette tip (diameter ~1.2μm).

[0083] The above content is only a specific embodiment of the present invention. It is also possible to select corresponding fluorescent labels (quenchers) for other ion channels (ion pumps, pore structures, and membrane proteins with similar properties), and use other alternative test instruments to synchronously record transmembrane electrical signals and protein fluorescence signals, and analyze the relationship between membrane protein function and structure. For those skilled in the art, based on the concept of the present invention, it is easy to make some changes or adjustments to certain specific embodiments and application scopes, and the contents of this specification should not be understood as limiting the present invention.

Claims

1. A fluorescence microscope, comprising: a laser that emits a parallel light beam in a specified direction; a beam shaping component, the beam shaping component being arranged on a laser emission path of the laser to form the parallel light beam into a parallel annular light beam; a beam guiding assembly disposed on a laser emission path of the laser; and objective lens; The beam guiding assembly guides the annular beam to the objective lens and focuses it on the imaging focal plane of the objective lens to form a focal spot. The focal spot is focused on the sample to be observed to generate a light signal, and the light signal is imaged through the objective lens and the beam guiding assembly. 2 . The fluorescence microscope according to claim 1 , wherein the axis of the beam shaping assembly coincides with the axis of the laser.

3. The fluorescence microscope according to claim 1, wherein the beam shaping assembly comprises: A convex lens, an aperture and a concave lens; the axes of the convex lens, the aperture and the concave lens coincide with the axis of the beam shaping component, and are arranged in sequence along the incident direction of the light of the beam shaping component. The fluorescence microscope according to claim 3 , wherein the front focal planes of the convex lens and the concave lens are aligned. The fluorescence microscope according to claim 1 , wherein the beam guiding component is a dichroic mirror. The fluorescence microscope according to claim 1 , wherein the laser is a continuous laser or a pulsed laser.

7. A method for combined detection of in situ single-molecule fluorescence and current signals using the fluorescence microscope according to any one of claims 1 to 6, comprising the following steps: Step 1: Select appropriate fluorescence imaging technology based on the detection requirements, and accordingly select the type of dye used for fluorescent labeling and the number of labels; use the selected dye to label the channel protein to be observed; Step 2: Assemble the channel protein onto the cell membrane surface and ensure that it can perform its normal function and the normal physiological process of the cell will not be significantly disturbed by the channel protein; the expression density of the channel protein is in the range of 0.1-10 μm 2 ; Step 3: Selecting a suitable activation signal to activate the channel protein; if the basal activity of the channel protein is 0, no activation is required; forming a single-channel recording area on the cell membrane surface and recording its electrical signal according to the single-channel recording patch clamp technical specifications; Aligning the single-channel recording area with the fluorescence imaging focal spot of the fluorescence microscope, performing single-molecule fluorescence signal imaging on the single-channel recording area with the fluorescence microscope, and simultaneously recording the fluorescence signal; Step 4: Analyze the fluorescence signal to extract the kinetic information of the channel protein; the electrical signal reflects the functional information of the channel protein; align the two in time to reflect the relationship between the kinetic information and the functional information of the channel protein.

8. The method according to claim 7, wherein in step 1: quantum dots or gold nanoparticles are used as markers instead of fluorescent markers.

9. The method according to claim 7, wherein in step 2: the channel protein is assembled onto the cell membrane surface by transfection, and is ensured to be able to perform its normal function. 10 . The method according to claim 7 , wherein in step 3: a trigger signal is set so that trigger information is included in the fluorescence signal record to ensure alignment between the two measurement signals.

11. The method according to claim 7, wherein step 3 comprises: Step 301, adjusting the fluorescence microscope to align the region of the channel protein to be observed with the focal spot; Step 302 , according to the single-channel recording patch clamp technical specifications, move the pipette tip to the area to be observed; Step 303, applying a test voltage according to the single-channel patch clamp technical specifications, lowering the pipette to form a single-channel seal at its tip, with an impedance greater than 10 GΩ, and forming the single-channel recording area; Step 304, start single-channel recording and remove the test voltage; Step 305 , adjusting the fluorescence microscope, selecting weak laser excitation, and adjusting the objective lens so that the signal to be observed at the tip of the pipette is clear; Step 306, adjusting the fluorescence microscope, selecting strong laser excitation, applying a channel protein activation signal, and completing the recording of the fluorescence signal and the electrical signal; The single-channel recording area formed in step 303 may not include the target single channel. If this happens, the process should return to step 301. 12 . The method according to claim 11 , wherein the fluorescence information recording instrument in step 306 is an EMCCD or a single photon detector.

13. The method according to claim 11, wherein the channel is a voltage-gated ion channel, and an appropriate voltage signal is selected to drive the channel; or the channel is a mechanical force-sensitive ion channel, and the application of mechanical force is controlled to coordinate the activation.

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