Total internal reflection fluorescence microscope

Total internal reflection fluorescence microscopy ( total internal reflection fluorescence english microscopy, TIRFM ) is a special method of light microscopy, in which the fluorescence of a drug over an evanescent ( decaying ) field is stimulated. To generate the evanescent field of light at the boundary of the cover glass is totally reflected to the drug. The restriction to observations close to the cover glass has TIRF Mikroskokopie together with interference reflection microscopy.

Construction and application

In TIRFM is used that light that falls at a shallow angle on a glass - water interface is totally reflected. In the water behind the glass forms in this case, an evanescent field, ie a light field whose intensity decreases exponentially into the sample, with a typical penetration depth for visible light of 100-200 nm being there fluorescent molecules, the light of the incident wavelength can absorb, they will be excited to emit fluorescence light. This leads to a very good limitation of the fluorescence generated in glass regions close to - the observable layer is 100-200 nm thin - so that a significantly better resolution along the optical axis ( z-direction) is obtained as the 500 nm at normal fluorescence microscopy or confocal microscopy. However, only the layer can be examined directly behind the glass.

It typically uses an inverted light microscope with an objective lens with oil immersion and very high numerical aperture, for example 1.45, in order to achieve a flat angle of incidence can. Either the excitation light at the edge of the lens is injected, so that it at a shallow angle to the cover glass drops (cis- TIRFM, left diagram ), or using an additional prism in which an evanescent field is formed (trans TIRFM, right diagram ). In the second implementation, the requirements of the lens are not so high, because there is no such a high numerical aperture is needed.

Application

The TIRF microscopy can be used for investigation of structures that are very close (about 200 nm for visible light) to a surface located. This can fluorescent molecules in the membrane of a cell or close to this (see picture above). In classical fluorescence microscopy, the near-surface signal would be masked by the background stray light. TIRF is particularly suitable for the study of exocytosis.

Using a suitable camera TIRFM is sensitive enough to detect single fluorescent molecules. The method is therefore also used to observe single -bonded to a glass surface molecules.

TIRFM can also in connection with fluorescence correlation spectroscopy ( FCS then referred to as TIR ), or single-particle tracking techniques are used to observe the dynamics of fluorescent molecules and quantified.