QVprep Biology Lessons

Principles of Microscopy



Microscopy: Microscopy is the technical field of using microscopes to view samples and objects that are not within the resolution range of the naked eye.

Basic types of microscopes: There are three well-known branches of microscopy: Optical, Electron and Scanning probe microscopy.

Principles of microscopy: Optical and electron microscopy involve physical phenomena like diffraction, reflection, or refraction of electromagnetic radiation interacting with the specimen followed by the collection of scattered radiation or another signal in order to create an image. This process may be carried out by wide-field irradiation of the sample as in the case of light microscopy and transmission electron microscopy or by scanning of a fine beam over the sample in confocal laser scanning microscopy and scanning electron microscopy. Scanning probe microscopy involves the interaction of a scanning probe with the surface of the sample of interest.

Basic mechanism behind optical microscopes: Optical or light microscopy involves passing visible light passing through or reflected from the sample through lenses for magnification. The resulting image can viewed or photographed.

Limitations of optical microscopes: Limitations of standard optical microscopy (bright field microscopy) lie in three areas; a) Optical microscope can only image dark or strongly refracting objects effectively b) Diffraction limits the resolution to approximately 0.2 micrometers c) Out of focus light from points outside the focal plane reduces image clarity

Staining: Live cells do not have sufficient contrast since the internal structures of the cell are colorless and transparent. The best way to increase contrast is to stain the different structures with selective dyes, but this often involves killing and fixing the sample. Staining may introduce artifacts which are apparent structural details that are caused by the processing of the specimen and are thus not really part of the sample under study.