Confocal total-internal-reflection fluorescence microscopy with a high-aperture parabolic mirror lens

We present a theoretical study of a new total-internal-reflection fluorescence microscope for the detection of fluorescence at a water- glass interface. The system is designed for confocal imaging and spectroscopy of nanoparticles and single molecules. Focusing and fluorescence collection through standard glass coverslips is accomplished by a parabolic mirror lens. The large aperture of the element is used to excite fluorescence within the evanescent field of a diffraction-limited focus and to collect focal emission with high efficiency. Tight focusing and supercritical excitation reduce the detection volume for fluorescent analyte molecules well below that of an attoliter (10(-18) L), which can be advantageous for monitoring surface binding of single molecules without interference from fluorescence of the unbound bulk. Calculations of the electric fields in the focus region and simulated confocal imaging demonstrate the performance of the system. (C) 2003 Optical Society of America.