A Phosphole Oxide Based Fluorescent Dye with Exceptional Resistance to Photobleaching: A Practical Tool for Continuous Imaging in STED Microscopy

Abstract

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.

Membrane‐Penetrating Carbon Quantum Dots for Imaging Nucleic Acid Structures in Live Organisms

“…STED bypasses this limit through deactivation of photoexcited dyes with ad epletion light beam (lateral resolution typically 50 nm). [12] Our photostable cQDs allowed 3D scans of nucleic acid structures,which often requires extended exposure to an intense laser (ca. [12] Our photostable cQDs allowed 3D scans of nucleic acid structures,which often requires extended exposure to an intense laser (ca.…”

Non-fused Phospholes as Fluorescent Probes for Imaging of Lipid Droplets in Living Cells

“…For cell imaging using phosphole probes, phosphate buffer saline pH: 7.4 (PBS) with DMSO as co-solvent have been employed earlier (Viry et al, 2008; Wang et al, 2015). Therefore, the excitation- and emission characteristics of the newly synthesized phospholes were measured both in pure DMSO and in PBS with 0.7% DMSO (PBSD) as co-solvent.…”

“…In these systems, the phosphole ring is fused with a phenyl- or naphtyl-ring. These types of probes have been used for imaging of HeLa cells, adipocytes, and preadipocytes (Wang et al, 2015; Yamaguchi et al, 2015). Herein, we synthesized a series of pyridyl- and thienyl-substituted phospholes (Scheme 1) and evaluated these compounds as fluorescent tools for fluorescent imaging of cells.…”

Merck–Banyu Lectureship Award: A. Fukazawa / Japan Prize: Y. Okamoto / Dr. Margaret Faul Award: S. Reisman

“…Her research interests include the development of novel functional organic materials, including fluorescent probes, NIR‐emissive fluorophores, dyes, and semiconductors, on the basis of physical organic chemistry and main‐group chemistry. Fukazawa was among the authors of a Communication on a phosphole‐oxide‐based fluorescent dye for continuous imaging and of a Communication on polycyclic pentalenes with enhanced Hückel antiaromaticity that was featured on the back cover of Angewandte Chemie .…”