Patch-clamped membranes have revolutionized the field of cellular electrophysiology, but nothing was known about their ultrastructure until they were imaged using the high-voltage electron microscope (HVEM). In present study we compute a tomographic three-dimensional (3D) reconstruction ofpatch-clamped membranes made from a Xenopus oocyte according to standard techniques. The sample was prepared for viewing in the HVEM according to the dry-mounting technique previously described. A tilt series was collected with a 2° angular interval and an angular range from -64° to 66°. All micrographs were recorded at 1 MV (Fig. 1). Our alignment procedure was hampered by the lack of good fiduciary markers and the presence of the wall of the micropipette, however, we were able to compute a preliminary reconstruction of reasonable quality using the weighted back projection method.Tomograms from the 3D reconstruction demonstrate that the cylindrical axis of the micropipette was not coaxial with the tilt axis of the HVEM state (Fig. 2). The reconstruction was rotated until it was coaxial with the y-axis of the reconstruction volume and a series of cylindrical projections were computed (Fig. 3). The projections from the inner radii show the thin profile of the disk which spans the micropipette, while the projections at larger radii show the distribution of features along the wall or the micropipette. The blurred area in the middle (and at the ends) of each cylindrical projection is caused by the missing angular range, while the blank area in the longest projection is a result of the slightly elliptical cross-section of the micropipette. A small 3D volume containing the pipette-spanning disk was extracted from the reconstruction volume (Fig. 4); the fibrous nature of the disk is evident. A different view of the disk is presented by removing tomograms which contain interfering features from the front and back walls of micropipette from the reconstruction in its original orientation (Fig. 5).
