Total internal reflection fluorescence microscope

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A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nm, can be observed.

Background

In cell and molecular biology, a large number of molecular events in cellular surfaces such as cell adhesion, binding of cells by hormones, secretion of neurotransmitters, and membrane dynamics have been studied with conventional fluorescence microscopes. However, fluorophores that are bound to the specimen surface and those in the surrounding medium exist in an equilibrium state. When these molecules are excited and detected with a conventional fluorescence microscope, the resulting fluorescence from those fluorophores bound to the surface is often overwhelmed by the background fluorescence due to the much larger population of non-bound molecules.

Solution

To solve this problem, the TIRFM was developed by Daniel Axelrod at the University of Michigan, Ann Arbor in the early 1980s. A TIRFM uses evanescent waves to selectively illuminate and excite fluorophores in a restricted region of the specimen immediately adjacent to the glass-water interface. Evanescent waves are generated only when the incident light is totally reflected at the glass-water interface. The evanescent electromagnetic field decays exponentially from the interface, and thus penetrates to a depth of only approximately 100 nm into the sample medium. Thus the TIRFM enables a selective visualization of surface regions such as the basal plasma membrane (which are about 7.5 nm thick) of cells as shown in the figure above. The selective visualization of the plasma membrane renders the features and events on the plasma membrane in living cells with high axial resolution.

TIRF can also be used to observe the fluorescence of a single molecule, making it an important tool of biophysics and quantitative biology.