The 2014 Gordon Research Conference on Three-Dimensional Electron Microscopy (3DEM) will be the 20th meeting of a series that began in 1985. This conference will present leading method development and biological research in three-dimensional structure determination of biological samples, from macromolecules to cells. The meeting will highlight recent progress in sample preparation, imaging, and data analysis. Progress in cellular and molecular electron microscopy will be presented, covering electron tomography, sub-volume analysis, single particle cryo-EM, and electron microscopy of ordered assemblies from helical, spherical or planar 2D crystal arrangements, among others. 3DEM has become a key method in structural biology and cell biology. Recent breakthrough developments in detector technology significantly improve signal-to-noise ratios of recorded images or movies. Improved data analysis algorithms allow dealing with smaller or heterogeneous particles, reaching vastly improved resolution. 3DEM is a unique technique for the analysis of structure and conformational dynamics of a wide variety of specimens ranging from macromolecules to macromolecular assemblies to viruses, organelles and whole cells in their native environment. A focus at the conference will be on technological advances in instrumentation, software development, data processing, and tools for interpretation and validation. Progress in correlative imaging methods, as well as new alternative approaches will be presented. Finally, this conference will demonstrate examples how 3DEM studies help elucidate biological function. The Conference will bring together the leading experts, junior researchers, and representatives of industrial partners. Following the tradition of previous conferences the program will offer an attractive combination of lectures and poster sessions. There will be two sessions with poster presenters selected for short talks. The program will also provide excellent opportunities for discussions and informal gatherings. Due to limited attendance (<200 participants) and the over-subscribed nature of previous meetings, early registration is encouraged.