Authors: D. Verdes, M. Rabe and S. Seeger
Affilation: University of Zurich, Switzerland
Pages: 545 - 548
Keywords: spectroscopy and imaging, bio sensors
We report a new two channel fluorescence microscopy technique for surface-generated fluorescence. The realized fluorescence microscope allows high resolution imaging of aqueous samples. The core element of the instrument is a parabolic mirror objective that is 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, is used for diffraction limited laser focusing and for collecting fluorescence at low angles with respect to the optical axis. By separated collection of the fluorescence emitted into supercritical and subcritical angles, two detection volumes strongly differing in their axial resolution are generated at the water/glass interface. The collection of supercritical angle fluorescence (SAF) (by the parabolic mirror) results in a strict surface confinement of the detection volume whereas collecting below the critical angle (by the aspheric lens) allows gathering the fluorescence emitted several microns deep inside the aqueous sample. Consequently, the signals from surface-bound and unbound diffusing fluorescent molecules can be obtained simultaneously.