A new tool based on two micromanipulators facilitates the handling of ultrathin cryosection ribbons

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.

Hierarchical imaging: a new concept for targeted imaging of large volumes from cells to tissues

“…The latest device published is designed for cryo-ultramicrotomy [24]. It consists of two micromanipulators, each offering a three-way movement.…”

Correlative light and electron microscopy methods for the study of virus–cell interactions

“…Artifacts including sample compression and crevassing of the surface are much more pronounced on vitreous sections compared to room temperature preparations . Development of methods continues in this area and may make the process easier in the future . For some samples this remains the only method which allows imaging in the most native state.…”

Electron microscopy for ultrastructural analysis and protein localization in Saccharomyces cerevisiae

“…While some proteins have been optimized to retain their fluorescence capacity after EM-preparation 132, successful approaches employed fixation by either HPF followed by CEMOVIS (see below, 133134135) or FS embedding in Lowicryl HM20 2114, or plunge-freezing before application of the Tokuyasu method 136. Alternatively, GFP can be indirectly localized on EM preparations through either immunolabeling or chemical reactions if the tag consists of GFP fused with an enzyme that generates an electron dense precipitate, such as in the FLIPPER tag 3.…”

“…Because of this limitation and the fact that CEMOVIS requires sophisticated equipment (HPF system, cryo-ultramicrotome, cryo-EM, cryo-holder for ET…) as well as a high degree of technical expertise, published works exploiting this approach are scarce but are steadily increasing especially due to the major availability in HPF technologies, which overcome the previous use of plunge or slam freezing for sample vitrification. So far yeast has been exclusively used for proof-of-principle demonstrations for methods to be applied with CEMOVIS 133134135, but the resolution degree shown is very promising. Another disadvantage of CEMOVIS is that frozen preparations cannot be immunolabeled, which limits localization studies.…”