Total Internal Reflection Fluorescence (TIRF) Microscopy

Total internal reflection (TIR) of an incident light beam occurs when light crosses an interface into a medium with a lower index of refraction at an angle larger than the critical angle. An evanescent electromagnetic field is generated close to the interface, with the intensity of the field decreasing exponentially with the distance.


z is the distance from the surface. d is the characteristic depth, which depends on the wavelength of the incident beam and the indices of refraction of the two media; typical values for d are in the range of 100 nm.

TIRF microscopy exploits the principle of TIR by selectively exciting molecules immobilized at a surface (in a range of ~100 nm) and reducing background signal from molecules in the solution.


For further information about TIRF microscopy:

-Daniel Axelrod; Traffic, 2001; 2: 764-774. “Total internal reflection fluorescence microscopy in cell biology“ pubmed citation

-Yasushi Sako, Takeshi Uyemura; Cell Struct. and Funct., 2002, 27: 357-365. “Total internal reflection fluorescence microscopy for single-molecule imaging in living cells“ pubmed citation

-an interesting website on TIRF microscopy: