In situ high-resolution cryo-EM reconstructions from CEMOVIS.

Area of Science:

• Structural Biology
• Cell Biology
• Microscopy Techniques

Background:

• High-resolution imaging of cells and tissues using cryo-electron microscopy (cryo-EM) requires electron-transparent samples (≤500 nm thick).
• Focused-ion-beam (FIB) milling is the standard for preparing cryo-EM samples (lamellae), but it involves material loss, limited imageable area, and ion beam damage.
• Cryo-electron microscopy of vitreous sections (CEMOVIS) presents a potential alternative to FIB milling for sample preparation.

Purpose of the Study:

• To evaluate CEMOVIS as an alternative to FIB milling for cryo-EM sample preparation.
• To assess the quality and imageable area provided by CEMOVIS.
• To demonstrate the feasibility of achieving high-resolution structural information from CEMOVIS samples.

Main Methods:

• Sample preparation using cryo-electron microscopy of vitreous sections (CEMOVIS).
• Analysis of vitreous sections, acknowledging potential damage like compression, shearing, and cracks.
• Application of two-dimensional template matching for structural reconstruction.

Main Results:

• CEMOVIS enables sectioning of samples in series, yielding significantly more imageable area compared to FIB-milled lamellae.
• Despite sectioning-induced damage, molecular structures within vitreous sections were largely intact in many regions.
• Reconstruction of the 60S ribosomal subunit from Saccharomyces cerevisiae cells at near-atomic resolution was achieved using CEMOVIS data.

Conclusions:

• CEMOVIS is a viable alternative to FIB milling for cryo-EM sample preparation, offering distinct advantages in terms of imageable area.
• The technique preserves molecular integrity sufficiently for high-resolution structural studies.
• CEMOVIS expands the possibilities for cryo-EM analysis of cellular structures, particularly for large-scale imaging studies.