USE OF FIB FOR PREPARATION OF FROZEN-HYDRATED SPECIMENS

点击翻译按钮获取中文摘要

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. + Marko, M., Hsieh, C., MoberlyChan, W., Mannella, C., and Frank, J. (2006) Focused ion beam milling of vitreous water: prospects for an alternative to cryo-ultramicrotomy. J. Microsc. 222(1)42-47. 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 general strategy for preparing the specimens was worked out, involving resolution of several technical 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) where 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. A major effort in the FIB project is to work out procedures for conveniently 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 a successful NIH Phase I and Phase II SBIR proposals, with us collaborators, and we have signed an non-disclosure document for this development work. A prototype 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 was designed to include 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.