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. Author manuscript; available in PMC: 2017 Aug 30.
Published in final edited form as: Cold Spring Harb Protoc. 2016 May 2;2016(5):pdb.top077800. doi: 10.1101/pdb.top077800

Figure 1.

Figure 1

Total internal reflection (TIR) microscopy. The proportion of refracted to reflected light can be changed when passing light through two different materials: one of higher refractive index and the other of lower refractive index. A critical angle can be reached that defines the point where all the light is refracted parallel to the boundary between the two mediums. Once this critical angle is passed, light is totally internally reflected and creates an evanescent wave that transmits into the second medium and decays exponentially. In total internal reflection fluorescence (TIRF) microscopy, this phenomenon is exploited to restrict illumination of fluorophores to only those within ∼100 nm of the coverslip (green spheres) while eliminating background excitation of fluorophores in solution (white spheres).