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 inherent difficulties associated with cryo-ultramicrotomy, we have begun 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 at the Harvard Center for Imaging of Mesoscale Structures, confirmed that vitreously-frozen water can be milled with the ion beam without devitrification: + Marko, M., Hsieh, C., MoberlyChan, W., Mannella, C., Frank, J. (2006) Focused ion beam milling of vitreous water: prospects for an alternative to cryo-ultramicrotomy. J. Microsc. 222(1) 42-47. This work represents a parallel line of development that avoids many of the technical difficulties associated with mechanical sectioning, including section compression, surface artifacts, and attachment problems. In order to demonstrate that biological material can be FIB-milled for subsequent cryo-electron tomography, we used plunge-frozen suspensions of bacteria on TEM grids. The bacteria (E. coli and cyanobacteria) were between 500 and 1000 nm in diameter, and the ice layer was in excess of 1000 nm in thickness. We cut the TEM grids in half under liquid nitrogen, and FIB-milled normal to the cut edge, thinning the frozen suspension to 200-500 nm. We performed electron tomography on several samples that were thinned to 500 nm in thickness. The specimens remained vitreous (based on electron diffraction), and the tomograms revealed no obvious signs of damage at the cut surface. + Marko, M., Hsieh, C., Schalek, R., Frank, J. and Mannella, C.A. (2007). Focused-ion-beam thinning of frozen-hydrated biological specimens for cryo-electron microscopy. Nature Methods: 4(3): 215-217. The major current effort in the FIB project is to develop convenient procedures for milling high-pressure frozen tissue for TEM tomography. We are collaborating with Hummingbird Scientific in this effort. This company, led by mechanical engineer Norman Salmon and materials scientist Eric Stach of Purdue, specializes in TEM and SEM specimen holders and stages. Hummingbird has written several successful NIH Phase I and Phase II SBIR proposals with our collaboration. Non-disclosure and cooperative research agreements are in effect for this development work. A prototype FIB cryo-stage system was purchased by the Resource in December 2006, and has been installed at the University of Albany's College of Nanoscale Science and Engineering on an FEI Nanolab V600 FIB/SEM instrument. The system includes special fixtures and equipment to take tissue samples from the high-pressure freezer, through the FIB, and into the TEM, while keeping the specimen below the devitrification temperature and free of frost at all times. During the last reporting period, M. Marko traveled extensively, giving invited talks on this new technique in biological cryo-EM. We have tested all the components of the Hummingbird cryo-FIB system, and suggested several modifications that were subsequently implemented. Our goal for this reporting period was to apply the technique to high-pressure frozen pelleted cells or blocks of tissue. We froze mammalian (RBC) cell pellets in custom high-pressure freezer carriers designed to fit in a TEM cryo-transfer specimen holder. To demonstrate that specimens can be vitreously frozen in the custom holders, we cut excellent frozen-hydrated cryo-ultramicrotome sections. As reported in our most recent paper on cryo-ultramicrotomy (Hsieh et al, 2006, J. Struct. Biol. 153:1-13), only the highest quality freezing can yield excellent frozen-hydrated sections.
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