Total Internal Reflection Fluorescence Microscopy
Total Internal Reflection Fluorescence Microscopy (TIRFM) exploits an induced evanescent wave in a limited specimen region immediately adjacent to the interface between two media having different refractive indices (typically the specimen and glass coverslip). This eliminates the background fluorescence from outside the focal plane. This significantly increases the signal-to-noise.
Refraction (or bending) of light occurs as it encounters the interface between two media having different refractive indices. Total internal reflection occurs when a collimated light beam encounters a boundary to a medium of lower refractive index. The light has to be at an angle of incidence greater than the critical angle. At this angle the refraction is parallel to the interface. At greater angles the light is reflected back into the first medium.
The reflected light generates an electromagnetic field adjacent to the interface, in the lower-index medium. This evanescent field is decays exponentially from the surface to a distance of a few hundred nanometres. Only fluorophores within this evanescent field are excited therefore reducing the background fluorescence. This dramatically increases the signal-to-noise allowing the detection of single molecules.
Chisty, L., Toseland, C.P., Fili, N. Mashanov, G.I., Dillingham, M.S., Molloy, J.E. & Webb, M.R. “Monomeric PcrA helicase processively unwinds plasmid lengths of DNA in the presence of the initiator protein RepD.” Nucleic Acids Research 2013 41:5010-5023. (Joint First Authors) ***NAR Featured Article
Toseland, C.P. & Webb, M.R. “Fluorescence nucleoside triphosphates for single molecule enzymology”. Methods in Molecular Biology 2011 778:161.
Fili, N., Toseland, C.P., Dillingham, M.S., Webb, M.R. and Molloy, J.E. “A single molecule approach to visualize DNA unwinding”. Methods in Molecular Biology 2011 778:193.
Fili, N., Mashanov, G.I., Toseland, C.P., Batters, C., Wallace, M.I., Yeeles, J.T.P., Dillingham, M.S., Webb, M.R. and Molloy, J.E. “Visualizing helicases unwinding DNA at the single molecule level”. Nucleic Acids Research 2010 38:4448.