Technology Development

The patch clamp technique (Neher, 1992) is the central method in modern electrophysiology. It allows the recording of single ion-channel currents, or alternatively currents from entire small cells (whole-cell recording). In traditional patch-clamping, a glass pipette is gently applied to the cell membrane as an "electrode" through control by a skilled operator; a slow and labor intensive process. Recent developments in planar patch-clamp technology has now made it possible to envision high-throughput patch-clamp systems where recordings can be made from 384 or more cells in parallel. A limiting factor in these systems is the size and cost of the amplifiers. By miniaturizing the entire patch-clamp system in silicon as an integrated circuit, both these problem can be resolved. We have designed, fabricated and tested the first fully integrated patch-clamp amplifier system using silicon-on-sapphire (SOS) technology. This project was a collaboration with Prof. Eugenio Culurciello.

Spherical reconstruction is a technique we are pursuing for the imaging of membrane proteins reconstituted into lipid vesicles. The idea is to exploit the geometry of small, spherical lipid vesicles both to aid in the determination of orientation angles and to allow compensation for the membrane density in electron micrographs.

Obtaining EM images of vesicles has been difficult because the preparation of the cryo-EM specimen involves a blotting step that removes nearly all vesicles from the EM grid. We have been developing an "affinity surface" that provides tethering sites for vesicles. This is an ultra-thin carbon film to which is adsorbed a low density of polyhistidine molecules. In the presence of Ni2+ the polyhistidines form tethering sites for vesicles doped with NTA lipids.

Cryo-EM specimens are typically supported by a carbon film containing holes on the order of 1 micron diameter. Best is to have uniform-sized holes in a regular array. We are developing a simple "rubber stamp" technique for patterning the holes. The stamp is a block of PDMS molded from a silicon micromachined master. It is used to transfer a patterned plastic film to a glass surface. The film is transferred to metal grids and forms a substrate for the evaporation of carbon.

We are working on algorithms for automatic particle selection in micrographs and for real-time image acquisition in the electron microscope. Our approach is a classical multi-channel matched filter. We make use of a reduced-dimension representation of the references for computational efficiency.