Breaking the resolution limit in light microscopy.

Abstract

Fluorescent imaging microscopy has been an essential tool for biologists over many years, especially after the discovery of the green fluorescent protein and the possibility of tagging virtually every protein with it. In recent years dramatic enhancement of the level of detail at which a fluorescing structure of interest can be imaged have been achieved. We review classical and new developments in high-resolution microscopy, and describe how these methods have been used in biological research. Classical methods include widefield and confocal microscopy whereas novel approaches range from linear methods such as 4Pi, I(5) and structured illumination microscopy to non-linear schemes such as stimulated emission depletion and saturated structured illumination. Localization based approaches (e.g. PALM and STORM), near-field methods and total internal refraction microscopy are also discussed. As the terms 'resolution', 'sensitivity', 'sampling' and 'precision' are sometimes confused, we explain their clear distinction. Key concepts such as the point spread function and the Abbe limit, which are necessary for an in depth understanding of the presented methods, are described without requiring extensive mathematical training.