Electron Microscopy and Three-Dimensional Image Reconstruction

This chapter focuses on the electron microscopy studies of Caenorhabditis elegans. The chapter discusses the methods for three-dimensional (3D) image reconstruction, based largely on film techniques introduced by John White and Randle Ware. Digital imaging techniques seem poised to make 3D reconstruction more accessible and may simplify the exchange of morphological data between laboratories. Several specialized specimen preparation techniques for electron microscopy, including freeze fracture and electron microscopic immunocytochemistry are described. The chapter outlines the methods making it feasible to collect hundreds of micrographs of cells or cell processes from a serially sectioned nematode. After assembling an ordered collection of negatives, all produced at one magnification, it is desired to view them sequentially in a well-aligned “movie” through the specimen. It can be quickly comprehended and can trace the continuity of individual processes as they course through a nerve bundle from a movie, even when the registration of profiles from print to print is difficult to discern.