Technique that utilizes total internal reflection of the incident light to generate a thin volume of excitation light at the reflection interface.
In vitro motility assay imaged with three color TIRF. Image courtesy of Melissa Hendershott and Ron Vale, Ph.D., UCSF
In TIRF (total internal reflection fluorescence) microscopy, excitation of fluorophores is confined to a very thin plane at the contact between the specimen and the cover glass. By confining excitation energy to this thin section, a high signal-to-noise-ratio is obtained, making it possible to observe single molecule fluorescence.
Due to its high signal-to-noise imaging property, TIRF is well suited for visualizing single molecules. As such, it is frequently used to assay behaviour of single molecules in reconstituted extracts. TIRF is also incredibly useful for studying endocytosis and exocytosis processes occurring at the plasma membrane as well as the dynamics of focal adhesions. Because TIRF imaging is limited to the cell-coverslip interface, it is not suitable for imaging structures that are more than ~200nm away from the interface.
TIRF can be configured on Nikon's Eclipse Ti series inverted microscopes.
Nikon Eclipse Ti series inverted microscope with environmental control, Nikon's dedicated TIRF Apo 60x NA 1.49 oil and TIRF Apo 100x 1.49 oil immersion objectives, and Nikon’s LUN laser unit with permanently aligned monolithic optics. The TIRF Apo objectives have correction collars which can be used to correct for temperature-dependent spherical aberration.