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

Authors: Watanabe, Takeshi1; Iketaki, Yoshinori2; Omatsu, Takashige3; Yamamoto, Kimihisa4; Fujii, Masaaki1

Source: Applied Spectroscopy, Volume 59, Issue 7, Pages 144A-160A and 853-963 (July 2005) , pp. 868-872(5)

Publisher: Society for Applied Spectroscopy

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Abstract:

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.

Keywords: FAR-FIELD FLUORESCENCE MICROSCOPY; SUPER-RESOLUTION MICROSCOPY; TWO-COLOR FLUORESCENCE DIP SPECTROSCOPY; TWO-POINT RESOLUTION

Document Type: Research Article

DOI: http://dx.doi.org/10.1366/0003702054411562

Affiliations: 1: Tokyo Institute of Technology, Chemical Resources Laboratory, 4259 Nagatsuta-cho, Midoriku, Yokohama 226-8503 Japan 2: Olympus Co., Ltd., 2-3 Kuboyama-cho, Hachioji, Tokyo 192-8512 Japan 3: Chiba Univ. Dep. Information & Imaging Science, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522 Japan 4: Keio Univ. Dept. of Chemistry, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522 Japan

Publication date: July 1, 2005

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