The Future of Patch Clamp Electrophysiology

Since the invention of the technique, voltage-clamp has relied on the use of glass microelectrodes that must be maneuvered onto a cell to form a gigaOhm seal with its membrane. There are great difficulties in maintaining a seal on a very small cell sandwiched between a glass slide on a microscope stage and a glass pipette attached to a mechanical micromanipulator anchored a great distance away. Vibrations in the system can reduce the success and longevity of an experiment. There are also limitations to the resolution of small currents due to the arrangement of an electrode being held high above a cell, acting as an antenna for electrical noise. Further, the process of approaching and forming a seal onto a cell with a micromanipulator is tedious and requires an operator with a high level of training. Finally, the fabrication of microelectrodes from glass tubes has not yet achieved a high level of reliability and therefore there is much variation in the results from use to use.

In recent years a number of companies have attempted to produce a planar microelectrode device that is capable of performing voltage-clamp experiments. Thus far, two of these companies have succeeded in producing such a product and will provide this new technology on the market in 2003. The approach involves making a micron-sized aperture on a planar substrate that is amenable to mass fabrication, and modifying the surface of the said substrate to produce seals on the cell membranes. Molecular Devices was the first to market with a device containing 48 such apertures. Their approach achieves a partial seal on the cell membranes of approximately 70% of the 48 apertures all done simultaneously, then makes use of an ionophore to gain sufficient electrical access to the cells to voltage-clamp up to 48 cells in parallel. Another company, Axon Instruments, a leading supplier of voltage-clamp amplifiers, has partnered with AVIVA Biosciences, a biochip developer, to provide an instrument that is capable of voltage-clamping up to 16 cells, simultaneously and in parallel. AVIVA's planar electrode chips achieve at least 90% gigaohm seals on the cell membranes, then gain electrical and fluid access to the cell by disrupting the patch of membrane bound within the aperture in much the same way as would happen with a conventional microelectrode. The latter instrument is able to control each of the 16 cells individually and intelligently, thereby avoiding wasted experiments. At present both companies are targeting the pharmaceutical industry, however products should be widely available shortly, making the voltage-clamp technique accessible to anyone.

Patch Clamping Biochip

Figure 3. The integration of patch clamp electrophysiology and intracellular Ca2+ fluorescence. Membrane currents are measured using headstage and patch clamp amplifiers coupled in series with the patch pipette. Cai+ indicators are introduced into the cell, either by inclusion in the pipette solution (shown in left upper inset) or by AM loading (see Fig. IB). Indicator molecules are excited by UV lamps or laser; the appropriate excitation wavelength for each dye is chosen using filters cubes or rotating filter wheels (shown here). The excitatory energy is transmitted to the dye-loaded cells using a mirrored filter and fluorescence is transmitted back through this filter from the cell. Emission is measured at pre-determined wavelengths selected, once again, by filter cubes or wheels (shown here). The fluorescence signal is quantified by CCD or photomultiplier tube (PMT) device for each wavelength. For dual excitation or emission dyes, the ratio of the two signals is recorded using a signal ratio processor. Current and Ca2+ signals are processed by computer software for simultaneous display.

Figure 3. The integration of patch clamp electrophysiology and intracellular Ca2+ fluorescence. Membrane currents are measured using headstage and patch clamp amplifiers coupled in series with the patch pipette. Cai+ indicators are introduced into the cell, either by inclusion in the pipette solution (shown in left upper inset) or by AM loading (see Fig. IB). Indicator molecules are excited by UV lamps or laser; the appropriate excitation wavelength for each dye is chosen using filters cubes or rotating filter wheels (shown here). The excitatory energy is transmitted to the dye-loaded cells using a mirrored filter and fluorescence is transmitted back through this filter from the cell. Emission is measured at pre-determined wavelengths selected, once again, by filter cubes or wheels (shown here). The fluorescence signal is quantified by CCD or photomultiplier tube (PMT) device for each wavelength. For dual excitation or emission dyes, the ratio of the two signals is recorded using a signal ratio processor. Current and Ca2+ signals are processed by computer software for simultaneous display.

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