Electrophysiology, measuring electrical current passing through single membrane channels in cells
The patch clamp technique (developed by Neher and Sakmann) uses a glass micropippete with a smooth open diameter of approximately 1µm. This contrasts with ‘sharp’ microelectrodes often used in other electrophysiology techniques. The pipette is filled with a solution dependent on the specific study or patch clamp technique being used. A metal electrode in contact with this solution conducts electrical changes to a voltage clamp amplifier. It is pressed against the cell membrane and suction is applied to suck membrane inside the pipette with the electrode to form an electrically tight ‘giga-ohm’ seal. In patch clamping a single electrode is used to patch clamp the cell membrane enabling the voltage to be maintained at a constant level while changes in current are recorded. Similarly the cell can be current clamped and changes in voltage recorded. A number of variations of the patch clamp technique can be performed including, for example:
Planar patch clamp: Instead of a glass pipette, a flat surface punctured with tiny holes is used
Cell-attached patch: The electrode remains sealed to the cell membrane while the pipette solution is altered (for example, by the addition of drugs) and resultant changes in activity recorded.
Inside out patch: Where the ‘sucked-in’ area of the membrane) is torn away from the cell and exposing the intracellular surface of the membrane – which in turn can be exposed to various media containing, for example, various intracellular ligands.
Whole cell patch: Where increasing suction ruptures the membrane and provides electrode access to the intracellular environment for the short time before the intracellular environment is diluted by the pipette contents.
The technique is used to study excitable cells i.e. those that produce a small electrical current when stimulated. These principally include nerve cells and muscle fibers.
Any electrophysiology application including patch clamping places great demands on the microscopy set up in terms of stability, suppression of vibration, isolation from electrical interference, room for manipulators and access to the specimen. Nikon’s FN1 physiology workstation has been especially designed to meet the demands of electrophysiology with streamlined manipulation and minimal noise.
In the design of the FN1, Nikon has minimised electrical noise by using fiber illumination to bring light into the system from outside the cage and by connecting ground pins to all main parts of the microscope. Vibration is minimised by rigid construction and reduced tremor when turning the nosepieces and magnification. The simple and slim I-shaped body provides more working space and better access around the microscope to position manipulators and other peripherals. The condenser, sub-stage and turret can be removed entirely from the FN1 body to allow for more free space, depending on the experiment. In addition, the height of the fixed stage can be easily and quickly changed by the user. Water dipping objectives are isolated from thermal and electrical transmission. The 16x objective has a wide 45º approach angle and 3.0mm long working distance for easy access and enhanced visibility. The combination of the 16x objective (with high N.A.) in combination with an intermediate magnification changer allows users to perform patch-clamping experiments without changing the objective. The FN1 can be used in combination with the A1+ confocal system. With the optional motorized Z-axis and enhanced A1 software, users can conduct images analysis, 3D slice imaging and reconstruction, and can obtain 3D image and electrophysiological data alternatively from the same specimen. Users can quickly switch between IR illumination and IR-DIC to ensure accurate recognition of micro needle tips and enhance flexible contrast. The microscope also allows users to select specific IR wavelengths for each specimen type:
• 700-750nm for high contrast images of thin slices with fine structures
• 800-900nm for high contrast images of thick slices with deep structures
• Off-center oblique IR illumination providing flexible contrast from various angles.