J Microsc. 2006 Apr;222(Pt 1):42-7.

Focused ion beam milling of vitreous water: prospects for an alternative to cryo-ultramicrotomy of frozen-hydrated biological samples.

Marko M, Hsieh C, Moberlychan W, Mannella CA, Frank J.

Source

Resources for Visualization of Biological Complexity, Wadsworth Center, Empire State Plaza, Albany, NY 12201, USA. marko@wadsworth.org

Abstract

The feasibility of using a focused ion beam (FIB) for the purpose of thinning vitreously frozen biological specimens for transmission electron microscopy (TEM) was explored. A concern was whether heat transfer beyond the direct ion interaction layer might devitrify the ice. To test this possibility, we milled vitreously frozen water on a standard TEM grid with a 30-keV Ga(+) beam, and cryo-transferred the grid to a TEM for examination. Following FIB milling of the vitreous ice from a thickness of approximately 1200 nm to 200-150 nm, changes characteristic of heat-induced devitrification were not observed by TEM, in either images or diffraction patterns. Although numerous technical challenges remain, it is anticipated that 'cryo-FIB thinning' of bulk frozen-hydratred material will be capable of producing specimens for TEM cryo-tomography with much greater efficiency than cryo-ultramicrotomy, and without the specimen distortions and handling difficulties of the latter.

PMID:: 16734713; [PubMed - indexed for MEDLINE]

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Research Support, N.I.H., Extramural

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RR01219/RR/NCRR NIH HHS/United States

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