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

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.