Basic Science Imaging Platform

Electron microscopy (EM) allows investigating objects of a size that cannot be resolved by using the light microscope. Although in the last decade technical advances in laser confocal microscopy (LSM) allowed to overcome the resolution limit of a light microscope (calculated by the formula of Abbe: approximately 1 µm), the fine structures of prokaryotic and eukaryotic cells and tissues, as well as viruses and molecules can only be visualized by electron microscopy. The ultrastructures of cell and tissue surfaces is investigated by scanning electron microscopy (SEM), while analyses of their internal structures require sectioned material and is investigated by using a transmission electron microscope (TEM). With special techniques, also macromolecules can be visualized by TEM. Atomic force microscopy (AFM) gives the opportunity to investigate molecular and sub-molecular structures.

The main advantage of EM is the high resolution; the main disadvantage is the necessity to investigate fixed or frozen material. Therefore, EM allows only obtaining “snapshots” of living material. The correlative microscopy tries to overcome this situation. Structures labeled with fluorochromes are identified in the fluorescence microscope or in the LSM, marked and selected for TEM investigation.


Brief technical description

Cells and tissues are either conventionally fixed with aldehydes and osmium tetroxide, dehydrated and embedded in epoxy or methacrylate resins, or immobilized by high pressure freezing and further processed by cryo-substitution and again embedding in resin (“state of the art”-technique). Resin blocs are cut using an ultramicrotome equipped with a diamond knife. Ultrathin sections (thickness: 70-80 nm) and semithin sections (thickness: 200-300 nm and more) are mounted on copper grids and viewed in the TEM. The semithin sections are used for electron tomography in order to obtain 3D-information of the fine structures of the respective specimen. (J Neumüller/M Pavelka, March 2012)