This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor 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. The results were presented at the Microscopy and Microanalysis meeting in 2005. 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 at Albany NanoTech (the University at Albany) will be fitted with a cryo-stage and cryo-transfer system, and provided with associated cryo-preparation equipment. In the previous reporting period, the experimental results on ice milling were published:+ 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. 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. A paper was written for Nature Methods, was revised after the initial review, and is now in the final review stage:+ Marko, M., Hsieh, C.-E., Schalek, R., Frank, J. and Mannella, C.A. (2006) Ion beam thinning of frozen-hydrated biological specimens for three-dimensional electron microscopy.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, consisting of mechanical engineers Norman Salmon and Mark Scheef, and materials scientist Eric Stach of Perdue, specializes in TEM and SEM specimen holders and stages. Hummingbird has written a successful NIH SBIR proposal, with us collaborators, and we have signed an non-disclosure document for this development work. A prototype system has been designed that 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. We have contracted with Hummingbird for delivery of this system, paid for by designated carried-over funds from the RVBC grant, contingent on the system meeting a detailed set of specifications that we have written. We expect first tests of the system at in December at Albany NanoTech. Our initial biological FIB results garnered considerable interest, and much time was spent traveling to present this work:+ Invited talk at Schloss Hohenkammer meeting on cryoultramicrotomy, Munich, Germany entitled 'Progress in electron tomography of ultramicrotomed and FIB-milled frozen-hydrated specimens.' May 22, 2006.+ Invited talk at the Cavendish Lab, Cambridge University, UK. entitled '3-D cryo-EM in biology: Specimen preparation and imaging for tomography.' May 26, 2006.+ Platform presentation at Microscopy and Microanalysis 2006 meeting, Chicago, IL, entitled 'Focused ion beam (FIB) preparation methods for 3-D biological cryo-TEM.' Aug. 1, 2006.+ Platform presentation at XVI International Microscopy Congress, Sapporo, Japan entitled 'Focused ion beam milling of frozen-hydrated specimens for 3-D cryo-TEM.' Sept. 5, 2006.+ Invited talk at Fourth International Congress on Electron Tomogrraphy entitled 'Progress in electron tomography of ultramicrotomed and ion-beam milled frozen-hydrated specimens.' Nov. 7, 2006.+ Invited talk at joint meeting of Dutch and Belgian electron microscopy societies, Lunteren, Netherlands entitled 'Electron tomography of frozen-hydrated specimens prepared by ultramicrotomey and focused ion beam milling.' Nov. 28, 2006.
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