No abstract

SummaryWe have recently reported electron tomographic studies of sections obtained from chemically fixed E. coli cells overproducing the 60-kDa chemotaxis receptor Tsr. Membrane extracts from these cells prepared in the presence of Tween-80 display hexagonally close-packed microcrystalline assemblies of Tsr, with a repeating unit large enough to accommodate six Tsr molecules arranged as trimers of receptor dimers. Here, we report the direct visualization of the Tsr receptor clusters in (i) vitrified cell suspensions of cells overproducing Tsr, prepared by rapid plunge-freezing, and (ii) frozen-hydrated sections obtained from cells frozen under high pressure. The frozenhydrated sections were generated by sectioning at − 150 ° C using a diamond knife with a 25 ° knife angle, with nominal thicknesses ranging from 20 to 60 nm. There is excellent correspondence between the spatial arrangement of receptors in thin frozen-hydrated sections and the arrangements found in negatively stained membrane extracts and plunge-frozen cells, highlighting the potential of using frozen-hydrated sections for the study of macromolecular assemblies within cells under near-native conditions.

No abstract

Using electron microscopy to localize rare cellular events or structures in complex tissue is challenging. Correlative light and electron microscopy procedures have been developed to link fluorescent protein expression with ultrastructural resolution. Here, we present an optimized scanning electron microscopy (SEM) workflow for volumetric array tomography for asymmetric samples and model organisms (, ,). We modified a diamond knife to simplify serial section array acquisition with minimal artifacts. After array acquisition, the arrays were transferred to a glass coverslip or silicon wafer support. Using light microscopy, the arrays were screened rapidly for initial recognition of global anatomical features (organs or body traits). Then, using SEM, an in-depth study of the cells and/or organs of interest was performed. Our manual and automatic data acquisition strategies make 3D data acquisition and correlation simpler and more precise than alternative methods. This method can be used to address questions in cell and developmental biology that require the efficient identification of a labeled cell or organelle.

SummaryA new oscillating cryo-knife for producing uncompressed vitreous sections is introduced. The knife is a modified cryo diamond knife that is driven by a piezo translator. Optimal setting for the oscillation was found to be in the inaudible frequency range of 20 -25 kHz. Yeast cells and polystyrene spheres were used as model systems to describe compression in the vitreous sections. We found that compression could be reduced by a factor of about 2 when the knife was oscillating. When the oscillator was turned off, sections were compressed by 40 -45%. However, only 15-25% compression was obtained when the knife was oscillating. In some cases completely uncompressed sections of yeast cells were produced. It was also found that the amount of compression depends on the specimen itself and on its embedding medium. With the results shown here, we demonstrate that the oscillating knife can produce high-quality vitreous sections with minimum cutting artefacts.

No abstract

A close to native structure of bulk biological specimens can be imaged by cryo-electron microscopy of vitreous sections (CEMOVIS). In some cases structural information can be combined with X-ray data leading to atomic resolution in situ. However, CEMOVIS is not routinely used. The two critical steps consist of producing a frozen section ribbon of a few millimeters in length and transferring the ribbon onto an electron microscopy grid. During these steps, the first sections of the ribbon are wrapped around an eyelash (unwrapping is frequent). When a ribbon is sufficiently attached to the eyelash, the operator must guide the nascent ribbon. Steady hands are required. Shaking or overstretching may break the ribbon. In turn, the ribbon immediately wraps around itself or flies away and thereby becomes unusable. Micromanipulators for eyelashes and grids as well as ionizers to attach section ribbons to grids were proposed. The rate of successful ribbon collection, however, remained low for most operators. Here we present a setup composed of two micromanipulators. One of the micromanipulators guides an electrically conductive fiber to which the ribbon sticks with unprecedented efficiency in comparison to a not conductive eyelash. The second micromanipulator positions the grid beneath the newly formed section ribbon and with the help of an ionizer the ribbon is attached to the grid. Although manipulations are greatly facilitated, sectioning artifacts remain but the likelihood to investigate high quality sections is significantly increased due to the large number of sections that can be produced with the reported tool.

No abstract