Lambert Instruments

TIRF (Total Internal Reflection Fluorescence) microscopy facilitates extremely high-contrast visualisation and thereby high sensitivity of fluorescence near the cover glass. This is done without disturbing cellular activity, thereby enabling tracking of biomolecules, and the study of their dynamic activity and interactions at the molecular level. TIRF enables the selective visualisation of processes and structures of the cell membrane and pre-membrane space like vesicle release and transport, cell adhesion, secretion, membrane protein dynamics and distribution or receptor-ligand interactions. The unique combination of TIRF and frequency domain FLIM makes it possible to measure lifetimes of for instance small focal adhesions near the cover glass.

High NA TIRF objectives (up to 1,49) make it possible to introduce illumination at incident angles greater than the critical angle () resulting in TIR (Total Internal Reflection) which is accompanied by the formation of an evanescent wave immediately adjacent to the coverglass-specimen interface. The evanescent wave reaches maximally a few hundred nanometers into the specimen and its energy drops off exponentially. For TIR to occur, the refractive index of the coverglass should be higher than the refractive index of the specimen (which is e.g. the case when using buffered saline solution).
The white-TIRF as well as the laser-TIRF system utilises this evanescent wave to excite fluorescent molecules in the thin section in contact with the coverglass (here: green dots). Because the specimen is not excited beyond the evanescent wave (here: white dots), this imaging system can produce fluorescence images with an extremely high signal-to-noise (S/N) ratio.

More links:
Wikipedia
Nikon Information Center
Olympus FluoView

At the TIRF-FLIM APPLICATIONS site, the difference between TIRF and widefield images of dSH2-GFP transfected cells (kindly provided by Ms. S.E. Le Devedec, Leiden University, The Netherlands) are shown. A photo of the TIRF-FLIM set-up can be found at the LIFA CONFOCAL site.