Confocal TIRF microscopy of single molecules

摘要

Total internal reflection fluorescence (TIRF) microscopy is a powerful technique to investigate surface bound emitters exclusively, i.e. with low interference of the fluorescing bulk in the adjacent aqueous solution. Confocal microscopy is a powerful technique to detect low emission intensities, like the emission of a single molecule, with high temporal resolution and high signal-to-noise ratio. We present a confocal total internal fluorescence microscope, which combines the virtues of diffraction limited confocal imaging and TIRF. Annular supercritical focusing and fluorescence collection through standard glass cover slips is accomplished by a parabolic mirror lens. Tight focusing and supercritical excitation reduce the detection volume for fluorescent analyte molecules well below attoliters. Beyond, the large aperture of the element leads to a high collection efficiency of surface bound emitters (～50%). The system is characterized in detail by calculations of the electric fields in the focus region and simulated confocal imaging. Single molecule experiments demonstrate the performance of the microscope in practice.