Parallel two channel near and far field fluorescence microscopy

Abstract

A two channel fluorescence microscopy technique for surface-generated fluorescence is presented. The realized fluorescence microscope allows high resolution imaging of aqueous samples. The core element of the instrument is a parabolic mirror objective used to collect the fluorescence at large surface angles above the critical angle of the water/glass interface. An aspheric lens, incorporated into the solid parabolic element, gives diffraction limited laser focusing and collects the fluorescence at low angles with respect to the optical axis. By separated collection of the fluorescence emitted into supercritical and sub critical angles, two detection volumes strongly differing in their axial resolution are generated at the surface of a glass cover slip. The collection of supercritical angle fluorescence (SAF) results in a strict surface confinement of the detection volume whereas collecting below the critical angle allows gathering the fluorescence emitted several microns deep inside the sample.