Structural analysis of multicellular organisms with cryo-electron tomography

Abstract

We developed a method for visualizing tissues from multicellular organisms using cryo-electron tomography. Our protocol involves vitrifying samples with high-pressure freezing, thinning them with cryo-FIB-SEM (focused-ion-beam scanning electron microscopy) and applying fiducial gold markers under cryogenic conditions to the lamellae post-milling. We applied this protocol to acquire tomograms of vitrified Caenorhabditis elegans embryos and worms, which showed the intracellular organization of selected tissues at particular developmental stages in otherwise intact specimens.

A focused ion beam milling and lift-out approach for site-specific preparation of frozen-hydrated lamellas from multicellular organisms

“…Thicker samples require substantially longer milling times (e.g. tens of hours, (Harapin et al, 2015)) and that inevitably will introduce damage to the final lamellas (data not shown). Thus, ''on the grid milling'' is neither efficient nor very promising for high-pressure frozen samples exceeding an initial thickness of 10 lm.…”

“…Thus, the development of cryo-FLM setups enabling higher resolution confocal imaging will be needed to solve these issues. Additionally, preparation of high-pressure frozen specimens in organic solvents that sublimate at temperatures below devitrification (Harapin et al, 2015), could assist in the production of thinner samples and subsequently lead to better imaging capabilities. Moreover, it could eliminate the need for cryo-planing to expose the biological sample at the surface prior cryo-FIB milling.…”

“…This procedure is advantageous for obtaining thin lamellas from a homogenous tissue, but would be difficult to apply for targeting specific sparse structures within it. Harapin et al recently reported an application of ''on the grid lamella milling'' to high-pressure frozen Caenorhabditis elegans worms and embryos (Harapin et al, 2015). However, the application suffers from the limitations described above for eukaryotic cells.…”

The assembly of C. elegans lamins into macroscopic fibers

“…In vitro, Ce-lamins assemble into native-like 10-nm filaments that are assembled from protofilaments. These filamentous structures are thought to exist within the nucleus (Harapin et al, 2015;Mahamid et al, 2016). Paracrystalline fibers, which are the major building blocks of the macroscopic fiber, are organized similarly to Ce-lamin 10-nm filaments.…”

Cellular structural biology as revealed by cryo-electron tomography

“…The first 3D-snapshots of the algal chloroplast were also obtained by this method, affording a glimpse into photosynthesis, thylakoid biogenesis and carbon fixation (Engel et al, 2015b). FIB milling and imaging by cryo-ET of frozen Caenorhabditis elegans embryos and adult worms has allowed researchers to visualize the intracellular organization during particular stages of development in tissues of interest (Harapin et al, 2015). Here, the milled lamellae were ∼330 nm thick in the case of embryos, and ∼660 nm thick for adult worms, both sufficiently thin to be visualized by cryo-ET (Fig.…”