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Two-Point Separation in Far-Field Super-Resolution Fluorescence Microscopy Based on Two-Color Fluorescence Dip Spectroscopy, Part I: Experimental Evaluation

Volume 59, Number 7 (July 2005) Page 868-872

Watanabe, Takeshi; Iketaki, Yoshinori; Omatsu, Takashige; Yamamoto, Kimihisa; Fujii, Masaaki


The two-point resolution of a novel two-color far-field super-resolution fluorescence microscopy was evaluated by measuring fluorescent beads 100 nm in diameter. This microscopy is based on a combination of two-color fluorescence dip spectroscopy and a phase-modulation technique for a laser beam. By simply introducing two-color laser light, the size of the fluorescent image of a bead was shrunk down to a diameter of 250 nm from the diffraction-limited image with a diameter of 360 nm. For two closely adjacent fluorescent beads with a separation distance of 350 nm, the two-color microscope clearly gave separated fluorescence images, while the conventional one-color fluorescence microscope could not resolve them. It has been proved that our technique breaks Rayleigh's diffraction limit.