TIRF Principles
TIRF in Theory and Practice
TIRF employs the phenomenon of Evanescent Wave (EW) that occurs at the interface between optically dense media such as glass (n=1.51) and optically less dense media e.g. water (n=1.33). At angles of incidence larger than the critical (62 degrees for glass/water interface) the excitation light reflects back into glass and generates EW at the glass/water interface (see the schematics). Maximum of intensity is at the glass surface. The intensity exponentially decays with the distance from the surface. The depth of penetration is ~100 nm. Only molecules at the surface and in close ~10 nm proximity to the surface are excited and fluoresce. Fluorophore in bulk of solution are not excited. In comparison, the confocal microscopy excites ~ 1,000 nm, while traditional methods such as epi-fluorescence illuminate the entire bulk of biological specimen, which generates large background fluorescence and an enormous scatter of light. The background masks useful fluorescence signals from the molecules of interest. TIRF efficiently minimizes the background and allows for supersensitive detection down to single molecules. Due to the superior spatial selectivity and exceptional sensitivity, TIRF has become the method of choice for single molecule detection, single molecule biology, and a number of super-resolution microscopy techniques that circumvent the diffraction limit and resolve single molecules. No other technique exists that can monitor fluorescence lifetime, polarization, anisotropy decay, quenching, resonance energy transfer (FRET), recovery after photobleaching (FRAP), and correlation spectroscopy (FCS) in real-time.
TIRF-ElectroChemistry (TIRF-EC) and TIRF- Electric Field
TIRF in combination with electrochemical, electric field, and dielectrophoretic control allows for the opportunity to monitor single molecules and simultaneously control their behavior, rotate and move them in XYZ dimensions. To perform a TIRF-EC experiment, the surface of the TIRF slide is coated with a transparent film of indium tin oxide (ITO), which is patterned to obtain several electrodes. In a typical TIRF-EC experiment, the electric field applied to central ITO electrode controls the behavior of molecules at and near the electrode, while TIRF monitors the behavior. Diagrams illustrate the principles of TIRF-EC. See Application Notes and request PDF reprints of TIRF-EC articles.
Chemically modified and bio-functionalized TIRF slides with reactive amine, epoxy, and other groups, biotinylated, and streptavidin-coated TIRF slides, and reagent kits for surface immobilization of biomolecules are available as consumables.
TIRF flow system used in conjunction with a fluorometer is well-suited for the analysis of biomolecular interactions, measurements of sensograms, the determination of k-on and k-off rate constants, and affinity constants K, for example, for antibody-antigen interactions. For this purpose, one of the partner of interactions, e.g. antibody, is immobilized at the surface of TIRF slide, while the other partner, e.g. antigen, is presented in the solution flow. The solution can be driven by gravity flow, which is always by hand, or by a TIRF flow system TA1004, that can be interfaced with digital fluidics SmartFlow TF1005, which transforms a fluorometer into a computer-controlled TIRF biosensor instrument. The sensogram is recorded, and k-on and k-off rate constants derived from the kinetic curve.
