TOTAL INTERNAL REFLECTION FLUORESCENCE MICROSCOPY (TIRF)

Total internal reflection fluorescence microscopy (TIRF) is a microscopy technique with which a thin region of the cell, usually less than 200nm can be observed. A TIRF microscope uses an evanescent wave to selectively illuminate and excite 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 internally 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 TIRF microscope enables a selective visualization of surface regions such as the basal plasma membrane (which are about 7.5 nm thick) of cells. This technique is often used also for observing molecular dynamics in vitro, or study the details of cell locomotion or adhesion to substrata.