Abstract
Three-dimensional reconstructions of large cellular subvolumes would be quite valuable in addressing a number of basic questions related to cell ultrastructure. More specifically, we are interested in obtaining reconstructions with several nm resolution over projected areas of 5-50 um diameter and 1-10 um depth. Recently, EM tomography has been combined with serial sectioning as a means of accomplishing this goal. In practice, however, a number of technical problems must still be solved to realize the full potential of this approach, and most applications of EM tomography to date have been limited to high resolution reconstructions over much smaller volumes or lower resolution reconstructions over larger volumes.A major limitation is the limited depth of focus of the electron microscope. Although this depth of focus usually exceeds the section thickness, at very high tilt the range of underfocus through overfocus in the image will approach the diameter of the reconstructed area.
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