A123 - A simple way to enhance TIRF microscopy using dedicated coverslips

Cyril Favard (cyril.favard@irim.cnrs.fr)

High numerical aperture objective based Total Internal Reflection Fluorescence Microscopy (TIRF-M) is widely used thanks to its simplicity of implementation (dedicated commercial microscopes) and its improved axial resolution, down to hundreds of nanometers. However, TIRF-M suffers from various limitations; in particular, its signal-to-noise ratio is intrinsically not sensitive and different sources of noise often make its optical contrast poor. An interesting solution is to increase the fluorescence signal without increasing the noise linked to the excitation laser, by acting on the evanescent field i.e. the fluorescence excitation field. In addition, although theoretically the evanescent excitation should appear at the critical angle, the angular divergence of the excitation filed due to the use of high NA objective is generating far field propagation, preventing the "TIRF" effect to be correctly achieved. This can be rescue by increasing the incident angle of excitation above the critical angle, however, this lead to strong decrease in fluorescence signal. To circumvent that, using dielectric multilayers (DM) we have designed coverslips that generate a strong evanescent field at high angle. These coverslips are fully biocompatible and reusable. The workshop will illustrate how using such coverslips on a commercial TIRF microscope strongly enhance the evanescent imaging.