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).
S upercritical
A ngle
F luorescence
U ndercritical
A ngle
F luorescence
Parallel 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).