A cylindrical specimen holder for electron cryo-tomography

Abstract

The use of slab-like flat specimens for electron cryo-tomography restricts the range of viewing angles that can be used. This leads to the “missing wedge” problem, which causes artefacts and anisotropic resolution in reconstructed tomograms. Cylindrical specimens provide a way to eliminate the problem, since they allow imaging from a full range of viewing angles around the tilt axis. Such specimens have been used before for tomography of radiation-insensitive samples at room temperature, but never for frozen-hydrated specimens. Here, we demonstrate the use of thin-walled carbon tubes as specimen holders, allowing the preparation of cylindrical frozen-hydrated samples of ribosomes, liposomes and whole bacterial cells. Images acquired from these cylinders have equal quality at all viewing angles, and the accessible tilt range is restricted only by the physical limits of the microscope. Tomographic reconstructions of these specimens demonstrate that the effects of the missing wedge are substantially reduced, and could be completely eliminated if a full tilt range was used. The overall quality of these tomograms is still lower than that obtained by existing methods, but improvements are likely in future.

Electron Tomography: A Three‐Dimensional Analytic Tool for Hard and Soft Materials Research

“…[111] FIB has also been utilized to prepare specimens of soft materials for 3D analysis that were previously difficult to analyze by TEM, [112,113] and even for improving reconstructions of frozen-hydrated cells. [114] …”

Progressive Stochastic Reconstruction Technique (PSRT) for cryo electron tomography

“…First, the specimen can be tilted only from roughly ±60° (Palmer and Loewe, 2014), leaving us with an incomplete set of projections (called a missing wedge problem). Second, the specimen is larger than a detector and thus some projections contain information about the specimen outside of the reconstructed area.…”

The promise and the challenges of cryo‐electron tomography

“…Computationally it is possible to infer what the missing wedge data would be, but it is not possible to recover unobserved data [161]. Obtaining tomograms over the full angular range has been successful for small bacteria trapped in glass capillaries [162]. To extend the angular range, circular FIB-milling patterns could be used to make cylindrical specimens at the expense of a much reduced volume.…”