The ultrastructure of patch-clamped membranes: a study using high voltage electron microscopy

We have developed techniques for studying patch-clamped membranes inside glass pipettes using high voltage electron microscopy (HVEM). To preserve the patch structure with the least possible distortion, we rapidly froze and freeze dried the pipette tip. The pipette is transparent for more than 50 microns from the tip. HVEM images of patches confirm light microscopy observations that the patch is not a bare bilayer, but a membrane-covered bleb of cytoplasm that may include organelles and cytoskeleton. The membrane that spans the pipette is commonly tens of micrometers from the tip of the pipette and occasionally as far as 100 microns. The structure of patches taken from a single cell type is variable but there are consistent differences between patches made from different cell types. With suction applied to the pipette before seal formation, we have seen in the light microscope vesicles swept from the plasmalemma up the pipette. These vesicles are visible in electron micrographs, particularly those made from chick cardiac muscle. Colloidal gold labeling of the patch permitted identification of lectin-binding sites and acetylcholine receptors. In young cultures of Xenopus myocytes, the receptors were diffuse. In 1-wk- old cultures, the receptors formed densely packed arrays. The patch pipette can serve, not only as a recording device, but as a tool for sampling discrete regions of the cell surface. Because the pipette has a constant path length for axial rotation, it is a unique specimen holder for microtomography. We have made preliminary tomographic reconstructions of a patch from Xenopus oocyte.