Electron Tomography of Frozen-Hydrated Sections

Abstract

Electron tomography provides a 3-D reconstruction derived from a series of projection images recorded as the specimen is tilted in the microscope. [1] Despite the challenges of low contrast and sensitivity to beam-induced specimen damage, electron tomography can be applied to frozenhydrated biological material. This avoids the necessity for chemical fixation, dehydration, and straining, and thus allows study of the specimen in a "near-native" state. A variety of specimens have been studied in aqueous suspension by electron tomography of thin films of vitreous ice prepared by rapid plunging into a cryogen. These include microorganisms and molecules [2], mitochondria [3,4], and the skeletal muscle triad junction. [5] We have succeeded in extending this 3-D imaging technique to frozen-hydrated tissue sections, which makes it possible to study a wide range of "native" sub-cellular structures in situ.The water in biological specimens must be frozen in "vitreous" or amorphous form in order to avoid nanometer-scale damage to the specimen due to ice crystal formation.[6] For tissue, the preferred method is high-pressure freezing, which greatly extends the depth to which good freezing can be obtained.[7] The frozen tissue must be maintained below the de-vitrification temperature (~ -140°C) throughout ultramicrotomy and microscopy. While the cutting of suitable frozen-hydrated sections is challenging, the collective experience of a few dedicated labs has led to improvements in instrumentation and technique that have greatly improved the success rate.[8] During recording of the tomographic tilt series, care must be taken not to exceed a total dose of about 5000 e -/nm 2 . This avoids serious distortions in the frozen sections, and prevents damage to the higher-resolution structure of the specimen. The use of automated procedures for recording the tilt series is important for limiting the cumulative electron dose. [9] In our experiments, freshly excised rat liver tissue was frozen in a Balzers HPC-110 high-pressure freezer. Specimen holders from the freezer were opened to expose the tissue and directly clamped in the microtome chuck. A Diatome diamond trimming knife was used to trim away the metal of the specimen holder and produce a block face 150 µm on a side. Sections 160-200-nm thick were cut at -160°C with a Leica UCT/ EM FCS cryo-ultramicrotome using a Diatome 35° "dry" cryo knife and a Diatome Static Line adjustable ionizer. After a few ribbons of sections had accumulated at the knife edge, the ionizer was switched off, and the sections were transferred to 200-mesh folding grids using an eyelash. One grid in the attached pair had a Formvar-carbon support film on which 20 nm colloidal gold particles were deposited, for use as alignment fiducials. Each grid was placed on a rectangular piece of indium foil, which was then folded to form an envelope and firmly pressed flat. The indium foil provided protection for the grid during handling and long-term storage under liquid nitrogen.[10]The indium foil envelopes were opened...