The study of membrane ion channel properties using the membrane clamp is an arduous, detailed and complicated work, which requires a high level of technical level and experimental conditions to ensure that the process of membrane clamp experiments is generally described.
Operation method
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Materials and Instruments
Cell Tissue Move The first step is to be drawn in two steps, the first one is 7-10mm long, with a diameter of less than 200μm, on which the second one is carried out, finally making the diameter of the tip 1-2μm, the purpose of the two-step drawing is mainly to make the taper of the front end of the electrode larger, and to shorten the length of the narrowness, so it can reduce the series resistance of the electrode, and also reduce the electrode liquid dialysis time during the whole-cell recordings. Since the diaphragm microelectrode is most avoided to be contaminated with dust and dirt, and even more avoided to touch the parts near the tip, it is generally required to be made before use.The second step is to coat the front end of the electrode with a silicone resin (sylgard), which is intended to reduce the capacitance between the electrode and the perfusate and to form a hydrophilic interface. After this treatment, the above capacitance can be reduced from 6 to 8 pF to less than 1 pF. Silicone resin has no effect on the formation of Giga?Jan seals, but reduces background noise, which is important for single-channel recordings. Satisfactory results can be obtained without the use of silicone resin when performing whole-cell recordings, and the microelectrodes are usually polished after applying the silicone resin, but it is preferable to polish them within one hour of application, otherwise it is difficult to change the shape of the electrode tip.The third step is polishing, the electrode will be fixed in the microscope table, under the mirror will be close to the tip of the heating wire, when the heating is energized, the tip of the electrode can be seen slightly retracted, at this time, the electrode becomes smooth, and the tip of the impurities burned away, to get a cleaner surface. This is conducive to close sealing with the cell membrane, and easier to maintain stability after sealing.The electrode should be filled with electrode liquid before the experiment, and because the electrode tip is thin, the liquid inside the electrode should be filtered with 0.2 μm filter membrane before filling. Generally, the electrode filling can be divided into two steps: tipfilling and backfilling. The tip of the electrode can be immersed in the inner liquid for 5s, due to capillary action of the solution will enter the tip of the electrode, and then from the back end of the electrode with a small polypropylene injection tube inserted near the tip of the solution filled to 1/4 length, and then gently popped off the tip of the residual bubbles can be removed by the finger. The resistance of the electrode after filling is generally 2 to 5 MΩ, while whole-cell recording is preferably 2 to 3 MΩ.3. Diaphragm Clamp Experimental System Depending on the electrophysiological requirements, different experimental systems can be set up, but there are a number of basic components in common, including mechanical components (anti-vibration bench, shielding, instrumentation racks), optical components (microscope, video monitor, monochromatic light system), electronic components (diaphragm clamp amplifiers, stimulators, equipment for data acquisition, computer system), and micromanipulators.In most membrane clamp experiments, good mechanical stability of all instruments and equipment is required to minimize the relative motion between the microelectrodes and the cell membrane. The shock-resistant table holds the inverted microscope and its fixed micromanipulator, while the rest of the equipment is placed outside the table. The shielding is made of copper wire mesh and is grounded to prevent interference with the probe circuits of the diaphragm clamp amplifier by stray electric fields from the surrounding environment. Instrumentation racks should be located close to the bench to facilitate the mating of measuring instruments with optical instruments.Inverted microscope It is the main optical component of the membrane clamp experimental system, which not only has better visualization and facilitates the contact of the glass electrode with the top of the cell, but also is used to achieve focusing with the help of a moving objective lens, which has better mechanical stability. The video monitor is mainly used to monitor the operation during the experiment, especially to be able to correspond the sealing parameters (e.g. sealing impedance) to the morphology of the cells for good sealing.The diaphragm clamp amplifier is the core of the whole experimental system, which can be used for single-channel or whole-cell recording, and its working mode can be voltage clamp or current clamp. In principle, the probe circuit of the membrane clamp amplifier, i.e. the I-V converter, has two basic structural forms, i.e. the resistive feedback type and the capacitive feedback type, the former is a typical structure, and the latter is particularly suitable for ultra-low-noise single-channel recordings because of the use of feedback capacitors instead of feedback resistors, which reduces the noise. As for the membrane clamp experiment of special computer hardware and the corresponding software programs appear one after another, so that the membrane clamp experiment easy to operate, improve efficiency. Such as with the EPC-9-type membrane clamp amplifier (containing ITC-16 data acquisition / interface card) supporting the use of software PULSE/PULSEFIT, which can produce stimulus waveforms, control data acquisition, but also analyze the data, and at the same time has a lock-in amplifier for membrane capacitance monitoring, a variety of software features integrated into one! For more product details, please visit Aladdin Scientific website.
Silicone resins Electrode fluids
Membrane microelectrodes Filter membranes Shock-resistant benches Shielded covers Instrumentation racks Monochromatic light systems Diaphragm clamp amplifiers Stimulators Equipment for data acquisition Micromanipulators