Visualization of Organelles In Situ by Cryo-STEM Tomography.

Area of Science:

• Structural Biology
• Cell Biology
• Microscopy

Background:

• Cryo-electron microscopy (cryo-EM) determines near-atomic resolution structures of biological macromolecules.
• Conventional cryo-EM tomography is limited by specimen thickness, necessitating sample milling which disrupts 3D context.
• Scanning transmission electron microscopy (STEM) offers high resolution but requires careful handling of sensitive biological samples.

Purpose of the Study:

• To present a protocol for cryo-tomography using STEM to overcome thickness limitations in cryo-EM.
• To describe microscope configurations and automation for cryo-STEM tomography.
• To demonstrate the application of cryo-STEM tomography for imaging cellular structures.

Main Methods:

• Developed a cryo-tomography setup utilizing scanning transmission electron microscopy (STEM).
• Described microscope configurations (two- and three-condenser systems) and automation using SerialEM software.
• Implemented enhancements for batch acquisition and correlative alignment with fluorescence microscopy.

Main Results:

• Successfully reconstructed a mitochondrion, visualizing its membranes, granules, and surrounding cytoplasmic components.
• Demonstrated the capability to image organelles within the cytoplasm and the nuclear periphery of adherent cells.
• Achieved high-resolution imaging without the need for focused ion beam milling.

Conclusions:

• Cryo-STEM tomography circumvents the thickness limitations of conventional cryo-EM.
• This technique provides detailed insights into the spatial organization of cellular organelles and macromolecular complexes.
• Cryo-STEM tomography is a powerful tool for studying cellular architecture in situ.