TIRF Principles
TIRF employs the phenomena of total internal reflection and Evanescent Wave (EW) that occur 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) with maximum intensity at the glass surface and exponential decay with the distance from the surface. The depth of EW penetration is ~10-100 nm. This is superior spatial selectivity. In comparison, the confocal microscopy excites ~ 1,000 nm. 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. In TIRF, molecules of interest should be located at or near the surface. Due to the fact of superior spatial selectivity and exceptional sensitivity, TIRF has become the method of choice for single molecule detection. Using TIRF resembles viewing stars at night; unlike an attempt (similar to epi-fluorescence) of viewing them under sunlight. TIRF switches off the background like night switches off the sunlight. Single molecules, like stars at night, become easily detectable.
The phenomena of TIRF and EW allow to circumvent the diffraction limit and resolve single molecules with sizes smaller than the wavelength, which enables several super-resolution microscopies. 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 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.
TIRF-ElectroChemistry (TIRF-EC) System
Electrochemical, dielectrophoretic, and temperature control are available as options for our TIRF spectroscopy and microscopy systems. 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. 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, and the photo above shows the TIRF-EC flow system installed at the sample compartment insert of Horiba JY SPEX Fluorolog fluorometer. See Application Notes and request PDF reprints of TIRF-EC articles.
