Fluorophores emission directions dependent on their distance to the surface of the glass coverslip (see Fig.). When a fluorophore is close to an interface its near field can becomes propagative. This emission appears into the glass in directions beyond the critical angle. Thus, while the subcritical emission is identical whatever the distance fluorophore / glass, only the fluorophores located in the immediate vicinity of the interface have such supercritical angle fluorescent (SAf) emission. This light sometimes referred to as “forbidden light” may represent up to 50% of the detected emission of the fluorescent emitter. This supercritical emission decreases very rapidly with the axial distance to the interface and provides an absolute axial nanometric location of fluorophores.
To take advantage of SAF emission, we use commercial high NA objective combine with pupil engineering approach . Only the detection path of the microscope is modify to isolate or modulate the SAF emission. It is thus straightforward to combine with most fluorescent microscope. So far we demonstrate the coupling with conventional epi-fluorescence, confocal , and dSTORM microscopes. Association with STED is currently on-going.