Three-Dimensional Electron Microscopy. A Gateway to Photosynthetic Structure

Abstract

Three-dimensional electron microscopy techniques have emerged as powerful and practical tools in cell and structural biology. These tools carry the potential of unlocking many novel details of cytoplasmic order and macromolecular structure with nanometer resolution. Interfacing this plethora of structural information with genomic and physiological data holds the promise of unprecedented advancement in biology and medicine. Three-dimensional electron microscopy techniques accomplish the generation of a three-dimensional dataset that represents overall shape as well as internal structure of an object. This is achieved by processing transmission electron microscopy (TEM) images at different viewing angles. The resulting dataset can be used to generate random (virtual) sections through the reconstructed volume. One three-dimensional electron microscopy approach, electron tomography, has matured into a technique that is now routinely applied to a variety of biological questions with great success. Using plastic-embedded or ice-embedded samples, biological structures can be investigated at a resolution of 5–10 nm within their cellular context. A second three-dimensional electron microscopy approach, single particle electron microscopy, is pushing the obtainable resolution of isolated macromolecules into the sub-nanometer range, revealing tertiary and secondary structural arrangements. The combination of three-dimensional electron microscopy techniques with cryo-preservation and imaging at cryogenic temperatures is revolutionizing structural biology. This combination permits the investigation of hydrated structures, and thereby allows a direct examination of biological structure, which closely resembles the in vivo state. This chapter gives an introduction into theoretical background of tomography, reconstruction, image formation by transmission electron microscopy and cryo-preservation. The two methodologies, electron tomography and single particle electron microscopy are discussed with detailed technical information. An example for each technique is presented.