Total internal reflection fluorescence microscope
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A total internal reflection fluorescence microscope (TIRFM) is an apparatus with which a thin region of a specimen (usually less than 200 nm) can be observed.
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.
To solve this problem, the total internal reflection fluorescence microscope (TIRFM) was developed. TIRFM elicits an evanescent wave that selectively illuminates and excites fluorophores in a restricted region of the specimen immediately adjacent to the glass-water interface. The evanescent wave is generated only when the incident light is totally reflected from the glass/water interface at a specific critical angle, so the specimen undergoes an electromagnetic field to a depth of only in approximately 200 nm from the surface. The fluorescence excitation intensity outside this field decreases exponentially with increasing distance from the surface, indicating that TIRFM enables a selective visualization of contact regions such as the basal plasma membrane of cells as shown in the figure below. The selective visualization of the plasma membrane renders the features and events on the plasma membrane in living cells with high spatial resolution.