This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: Because of the inherent difficulties associated with cryo-ultramicrotomy, we are engaging in a highly novel, parallel approach to generate thin specimens of bulk biological tissue and whole cells: the use of a focused ion beam (FIB) to mill the specimens. The first experiments, carried out with Dr. Warren MoberlyChan at the Harvard Center for Imaging of Mesoscale Structures, confirmed that vitreously-frozen water can be milled with the ion beam without devitrification. Next, a general strategy for preparing the specimens will be worked out, involving resolution of several technical problems, all of which appear to be tractable. This represents a parallel line of development that, in principle, would avoid many of the technical difficulties associated with mechanical sectioning, including section compression, surface artifacts, and attachment problems. A dual-beam FIB/SEM in the Albany area will be fitted with a cryo-stage and cryo-transfer system, and provided with associated cryo-preparation equipment.
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