Principles of evanescent wave microscopy
TIRF microscopy, or evanescent field microscopy, is a form of fluorescence microscopy where the excitation light is confined to a small area at the interface between the sample and the glass slide or culture tank. The TIRF technique takes images of molecules isolated at the nanometric level. Numerous cellular processes make the cell’s plasmic membrane intervene: the formation and stabilisation of adherence areas, membrane receptor traffic, fusion of vesicles or their granulation, or recruitment of proteins on the plasmic membrane. These phenomena may of course be studied using confocal microscopy but soon the question of knowing whether or not the marker observed is found in close proximity to the membrane or on the membrane arises. An evanescent wave system, also called TIRF microscopy (Total Internal Reflection Fluorescence), may help clarify these problems and also help make significant improvements in axial resolution, speed of acquisition, and limitation of cytotoxicity from illumination.
TIRF is based on the following principle:
When light passes through an environment with a high refractive index (for example, glass) to an environment with a lower refractive index (for example, water or cells), and hits the interface between these environments at a sufficiently low angle of incidence, it is impossible for it to propagate in the second material while also being reflected in the first. However, the reflected light penetrates the second environment (cells) to a depth of only a few hundred nanometres and it decreases exponentially with distance. Excitation of the fluorophores therefore takes place in an area where the thickness is typically less than 200 nm. In confining the excitation energy in this thin section, a high signal to noise ratio is obtained, and it becomes possible to observe the fluorescence of individual molecules.
From refraction to total internal reflection.
(A) When a polarised monochromatic beam of light (laser) illuminates an interface between two environments with different refractive indices (for example, the interface between a glass slide and a cell), part of the incident light is reflected on the interface and the other part of the light is refracted through the surface.
(B) Depending on the beam’s angle of incidence, all of the light may be reflected. In conditions of total reflection, one of the electromagnetic components of the light, the evanescent waves, propagates perpendicularly to the interface with an intensity that decreases exponentially with distance from the interface.
By using TIRFM it is also possible to see and study the morphology or the events occurring at the plasmic membrane of living cells very selectively and at a very high resolution.