Supercritical Angle Fluorescence (SAF) microscopy is a versatile technique to study fluorescence species (biomolecules, nanoparticles, membranes etc.) and elucidate structures and dynamics at interfaces and on membranes. The detection volume in SAF microscopy can be confined to the first few hundred nanometers above the cover glass allowing for the selective observation of the surface-near region even in presence of high fluorophore concentrations deeper inside the specimen. SAF microscopy can be performed simultaneously with confocal microscopy allowing to also visualize deeper regions of a specimen. Theoretical background: SAF microscopy is based on the anisotropic emission of fluorescence near interfaces. Only emitters within about a wavelength distance to the glass emit a substantial proportion of light above the critical angle and by exclusive collection of the supercritical angle fluorescence the detection volume is confined to the surface. Applications: The excitation beam is focused to a tight spot of Gaussian intensity profile, ideal for combination with fluorescence lifetime imaging microscopy (FLIM), fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP) and multi photon excitation (MPE). SupercriticalAngleFluorescenceUndercriticalAngleFluorescenceParallel SAF microscopy (left) and confocal microscopy (right) of a cell microtuble network.
Microscope optics: The implementation of SAF microscopy on common inverted microscopes is facilitated by a new microscope objective. The optics features a diffraction limited focusing performance throughout the visible spectrum. It is designed for use with conventional glass coverslips (n = 1.52, d = 0.17 mm).