Preparation of cryofixed cells for improved 3D ultrastructure with scanning transmission electron tomography

Abstract

Scanning transmission electron tomography offers enhanced contrast compared to regular transmission electron microscopy, and thicker samples, up to 1 μm or more, can be analyzed, since the depth of focus and inelastic scattering are not limitations. In this study, we combine this novel imaging approach with state of the art specimen preparation by using novel light transparent sapphire specimen carrier for high-pressure freezing and a freeze substitution protocol for better contrast of membranes. This combination allows for imaging membranes and other subcellular structures with unsurpassed quality. This is demonstrated with mitochondria, where the inner and outer mitochondrial membranes as well as the membranes in the cristae appear in very close apposition with a minimal intermembrane space. These findings correspond well with old observations using freeze fracturing. In 880-nm thick sections of hemophagocytes, the three-dimensional structure of membrane sheets could be observed in the virtual sections of the tomogram. Microtubules, actin and intermediate filaments could be visualized within one sample. Intermediate filaments, however, could even be better observed in 3D using surface scanning electron tomography.

Self-pressurized rapid freezing (SPRF) as a simple fixation method for cryo-electron microscopy of vitreous sections

“…3B). This is in agreement with previous results on healthy and fully energized mitochondria (Hohn et al, 2011), confirming a very 'close to native' structure preservation using SPRF followed by CEMOVIS. In yeast cells, we also found excellent cellular ultrastructure quality.…”

Direct Image-Based Correlative Microscopy Technique for Coupling Identification and Structural Investigation of Bacterial Symbionts Associated with Metazoans

“…However, other techniques may provide potential relevant improvements to this method. In particularly, cryofixation followed by freeze substitution is known to carry out a better preservation of biological tissues than chemical fixation and to reduce fixation artifacts (26). Quantum dots also appear to be an attractive option in the choice of fluorescent dyes, because of their fluorescent properties and electron density (49).…”

Recent progress in histochemistry and cell biology

“…Unfortunately, the thickness of samples used is limited especially due to chromatic aberration. Applying scanning transmission electron microscopical tomography Hohn et al (2011) improved the acquisition of three-dimensional data of sample layers up to 1 lm by optimizing sample preparation, using improved high-pressure freezing, plastic embedding and a novel freeze-substitution protocol. These amended methods led to an advanced visualization of 3D structures of membrane sheets in 880-nm-thick sections of hemophagocytes.…”