Laser-free TIRF microscopy in cell biology

This article introduces Total Internal Reflection Fluorescence Microscopy (TIRFM), an imaging system that is based on the total internal reflection of light rays at the boundary between the glass of the chamber and the culture or growth medium. TIRFM uses a mercury vapour lamp light source to excite fluorescence within only the first few nanometres of a cell preparation - known as an evanescent lamella of illumination - thus allowing the selective visualisation of the morphology and molecular composition of cellular adhesion areas. TIRFM is an excellent complement to conventional fluorescence microscopy, since it is compatible with established cell culture systems as well as the methods currently used for fixation and labelling. TIRFM provides many other advantages such as excellent signal-to-noise ratio - especially in thick preparations - and it allows the use of multiple fluorophores, which is very useful when studying complex adhesion interactions.