By employing a spatially restricted evanescent field for the excitation of fluorophores, TIRF microscopy allows observation of the localization and dynamics of molecules and processes in an optical section near the plasma membrane (usually around 100 nm). This is advantageous for many applications dealing with processes in or close to the plasma membrane.
TIRF microscopy is an excellent technique for combining kinetic studies with spatial information in live samples or even in vitro. It is routinely used for investigating molecule trafficking as it occurs e.g. in cytoskeleton assembly. The rapid image acquisition and the outstanding background elimination in TIRF microscopy provide superb conditions for observing dynamic events like the recruitment of proteins to the plasma membrane. Thus, the kinetics of dynein/kinesin-mediated transport can be investigated, for example. It is also possible to track whole organelles like mitochondria using TIRF microscopy. For investigations in cell-cell interaction, special structures like focal adhesions of cells can easily be visualized with TIRF microscopy to observe e.g. the recruitment of parts of the cytoskeleton to focal adhesions.
By combining mathematical models (e.g. centroid tracking methods) with the unparalleled signal-to-noise ratio and z-resolution of TIRF microscopy, subdiffraction-limited localization of single molecules is achievable with a precision of 1 nm. This is possible as the fluorophore excitation by an evanescent wave produces low background fluorescence from out-of-focus fluorophores, resulting in a low signal-to-noise-ratio in a defined volume of the sample (e.g. the penetration depth of the evanescent wave multiplied by the area of the field of view). In a conventional lamp-based fluorescence system all fluorophores in the beam path are simultaneously excited and detected without any information about their z-position.
In other words: the three dimensional distribution of the fluorophores is displayed in only two dimensions as the different z-planes of the cell appear as one plane in the acquired image. This results in overlaying of the fluorophores in the image, which often makes the discrimination of single fluorescence spots impossible. In TIRF microscopy, however, only a comparably small number of the fluorophores within the approximately 100 nm deep evanescent wave is excited, providing an optical z-section of the sample.
The spatial proximity between fluorescence spots in TIRF images in x- and y-direction is relatively low as the light emitted from fluorophores from other z-planes is not overlaying the detected signal. If mathematical models (e.g. centroid tracking methods) are then applied to calculate the center of mass of the detected fluorescing molecule, subdiffraction-limited localization of single molecules is possible with a precision of 1 nm.