33.8: Total Internal Reflection Fluorescence Microscopy
Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass. This happens as the sample has a lower refractive index than the coverslip and does not allow the light to enter within. The light is reflected from the interface and forms an electromagnetic field emitting short-length evanescent waves. These waves only excite the fluorophore near the surface as they can move only about 100 to 200 nm deep within the cell before dying out.
There are two types of TIRF; prism-based and objective-based. In prism-based TIRF microscopy, a prism is placed on the coverslip surface that directs the evanescent wave to the sample. In objective-based TIRF microscopy, there is no prism at the interphase; the objective is the same as the light source that helps create the evanescent wave.
TIRF has several advantages over traditional fluorescence and confocal microscopes; it prevents the illumination of background fluorophores. It helps in studying the structures close to the cell surface. It reduces the blurring effect and does not allow out-of-focus light to interfere with the image. As the samples are not directly exposed to an intense light beam, the photobleaching is minimum, and the cells are less exposed to phototoxicity.
