A fundamental hurdle in freezing biological tissues is the formation of ice crystals that distort or damage structures. Cryoprotectants can damage structures, such as membranes, or distort cells by shrinking them, but cryoprotectants can be avoided if the heat can be removed in one millisecond or less. To date it has only been possible to achieve these rates of cooling within a few micrometers of the surface in a neural tissues (Heuser and Reese, 1981 (link)), limiting investigation almost entirely to disassociated cultures or surfaces of acute brain slices. High pressure freezing (HPF) methods (Moore, 1987 ), which inhibit formation of ice crystals by exertion of large pressures at the moment of freezing, can extend the depth of freezing to 100–300 μm or more and, when optimally implemented, can well preserve structures down to atomic resolutions (Dubochet, 2007 (link)). However, in practice, results may be capricious and current HPF machines limit the size of the tissue to one to three millimeters. Other HPF methods to freeze reliably larger volumes of tissue are under development. The heat transfer rate for HPF is generally lower than that of rapid slam freezing (Heuser and Reese, 1981 (link)), so HPF might not be the best choice for capturing very fast dynamic events. Despite of these challenges, HPF would be the method of choice for preparing nerve tissues for many future EM tomography investigations where the emphasis is focused on macromolecular organization in neurons. Here we present an example of a system for tomography based on HPF and freeze-substitution of dissociated neuronal cultures (Fig. 1).