A Phosphole Oxide Based Fluorescent Dye with Exceptional Resistance to Photobleaching: A Practical Tool for Continuous Imaging in STED Microscopy.
The development of stimulated emission depletion (STED) microscopy represented a major breakthrough in cellular and molecular biology. However, the intense laser beams required for both excitation and STED usually provoke rapid photobleaching of fluorescent molecular probes, which significantly limits the performance and practical utility of STED microscopy. We herein developed a photoresistant fluorescent dye C-Naphox as a practical tool for STED imaging. With excitation using either a λ=405 or 488 nm laser in protic solvents, C-Naphox exhibited an intense red/orange fluorescence (quantum yield ΦF >0.7) with a large Stokes shift (circa 5900 cm(-1) ). Even after irradiation with a Xe lamp (300 W, λex =460 nm, full width at half maximum (FWHM)=11 nm) for 12 hours, 99.5 % of C-Naphox remained intact. The high photoresistance of C-Naphox allowed repeated STED imaging of HeLa cells. Even after recording 50 STED images, 83 % of the initial fluorescence intensity persisted.
Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Furo, Chikusa, Nagoya 464-8602 (Japan).
Chenguang
C
Wang
Department of Chemistry, Graduate School of Science, Nagoya University, Furo, Chikusa, Nagoya 464-8602 (Japan). aiko@chem.nagoya-u.ac.jp.
Aiko
A
Fukazawa
Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Furo, Chikusa, Nagoya 464-8602 (Japan). taki@itbm.nagoya-u.ac.jp.
Masayasu
M
Taki
Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Furo, Chikusa, Nagoya 464-8602 (Japan).
Yoshikatsu
Y
Sato
Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Furo, Chikusa, Nagoya 464-8602 (Japan). higashi@bio.naogya-u.ac.jp.|Division of Biological Science, Graduate School of Science, Nagoya University, Furo, Chikusa, Nagoya 464-8602 (Japan). higashi@bio.naogya-u.ac.jp.
Tetsuya
T
Higashiyama
Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Furo, Chikusa, Nagoya 464-8602 (Japan). yamaguchi@chem.naogya-u.ac.jp.|Department of Chemistry, Graduate School of Science, Nagoya University, Furo, Chikusa, Nagoya 464-8602 (Japan). yamaguchi@chem.naogya-u.ac.jp.
Shigehiro
S
Yamaguchi
D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D007834:Lasers; D008856:Microscopy, Fluorescence; D017553:Phosphorus Compounds / Q000737:chemistry*; D038761:Photobleaching
D005456:Fluorescent Dyes; D017553:Phosphorus Compounds
STED microscopy; fluorescent probes; phosphorus heterocycles; photophysics; photoresistance