Electron Microscopy

Differences between electrons and X-rays. The main electron microscopy techniques used in structural biology. The electron microscope: source, electromagnetic lenses, compustage, detector. Direct detection cameras and their characteristics. Physical principles of image formation in the microscope: "weak phase object" approximation, effect of the objective lens (astigmatism and spherical aberration), wave function in the back focal plane, image formation. Contrast Transfer Function (CTF) and its correction with phase flipping and Wiener filter. Phase plates.

Sample preparation: negative staining and cryo-EM. Sugar embedding. Sample preparation for single-particle cryo-EM. Electron microscopy techniques for high-resolution structural biology. Data collection: diffraction images and patterns. Electron crystallography. Preparation of two-dimensional crystals. Diffraction data collection: the reciprocal space of two-dimensional crystals. Analysis of diffraction data. Analysis of electron microscopy images of two-dimensional crystals: filtering, unbending, CTF correction and merging to obtain the projection map. Two examples of structures obtained with electron crystallography: bacteriorhodopsin and aquaporin-0.