A 360° single‐axis tilt stage for the high‐voltage electron microscope

Abstract

A new type of specimen stage that permits more than 180' of tilting about the axis of a side-entry rod has been developed for a high-voltage electron microscope (HVEM). Roughly cylindrical specimens, with radial dimensions of less than a few micrometres, that can be mounted on the tip of a microneedle or micropipette are applicable. For glass micropipettes, the energy of the 1-MeV beam of the HVEM is sufficient to image specimens through both walls. T h e stage employs a spindle mechanism that holds these needles or micropipettes coaxial with the tilt axis, allowing the specimen to be rotated without restriction. This arrangement, along with the cylindrical form of the specimen, is an important development for single-axis tomography, because it permits a complete 180" set of projections to be recorded. T h e angular accuracy of the stage was demonstrated to be within ? 0.20', with a cumulative error of less than 1.0" over a 180" span.The new stage was tested using puffball spores mounted on a micropipette. A 180" tilt series was recorded and processed to yield a tomographic three-dimensional reconstruction which was displayed both as a cross-sectional view perpendicular to the tilt axis, and as a shaded surface viewed from different directions. T h e same computations were repeated using subsets of the tilt series to assess the effect of various amounts of missing information. Visual inspection of a selected cross-section from these reconstructions indicated that limiting the angular range to 160" produced results nearly as good as the full data set. Limiting the range to 140°, however, produced a noticeable geometric distortion, which became increasingly severe with ranges of 120" and 100".

The internal compartmentation of rat‐liver mitochondria: Tomographic study using the high‐voltage transmission electron microscope

“…This goal will require improving current alignment schemes (in progress) as well as seeking ways to fill in the missing angular range of tilts. An approach to the latter problem is the development of cylindrical specimen holders for collection of images over the full 180" tilt range (Barnard et al, 1992). Finally, because of concerns regarding the effects of specimen preparative techniques on mitochondrial structure, low-denaturation and cryosubstitution protocols will be employed for optimizing structural preservation.…”

Micromachining tools and correlative approaches for cellular cryo-electron tomography

“…This might involve the fabrication of cooled micromanipulators for in situ 'lift out' procedures ( Fig. 5, similar to those known from TEM lamella preparations at room temperature) and the fabrication of cylindrical milling geometries that can be used in conjunction with rotation-tilt tomography holders, which in turn, do not suffer from an increase in specimen thickness during rotation, as experienced with a 'slab' geometry (Barnard et al, 1992;Kamino et al, 2004).…”

Site-specific 3D imaging of cells and tissues with a dual beam microscope

“…Thus, it is possible to generate planar or crosssectional thin sections, or even cylindrically symmetric specimens for electron tomography. Cylindrically symmetric specimens, mounted such that the long axis is oriented along the microscope tilt axis, can be superior to flat sections for purposes of structure determination using electron tomography, because the loss in vertical resolution due to the missing wedge effect can be largely eliminated (Barnard et al, 1992;Lee and Subramaniam, 2004). The capability of site-specific milling provides a potentially interesting alternative to the use of manually operated microtomes for the generation of sections for histological examination using either electron or light microscopy.…”