Reconstructing mammalian membrane architecture by large area cellular tomography.

Microscopy has provided crucial insights into the fundamental features and architecture of mammalian cells and organelles for now over a century. These glimpses of cellular fine structure have thus guided our impressions-as molecular cell biologists-of how mammalian cells and their numerous membrane-bound internal compartments are organized within three dimensions (3D), although for the most part these extrapolations have come from static two-dimensional (2D) images taken from the light or electron microscope. Recently, however, we have finally been afforded the chance to dissect subcellular membrane architecture and dynamics at the nanoanatomical level in 3D through development of the technique referred to as electron microscope (EM) tomography, also frequently termed "cellular tomography" (ET). With ET now in hand as a tool that effectively allows one to study molecular membrane traffic "in context" (in situ), it has become increasingly important to push the continued advancement of this method so that large cellular volumes can be reconstructed without sacrificing the ability to clearly resolve structures of interest. Likewise, it has become critical to do this as quickly and efficiently as possible in order to generate statistically significant sample sizes that offer reliable insights into cell organization under different physiological or experimental conditions. In this chapter, some of the technical developments, as well as key biological questions that have driven the development of large area ET, will be presented and discussed.