Near-Infrared Spontaneously Blinking Fluorophores for Live Cell Super-Resolution Imaging with Minimized Phototoxicity

Anal Chem. 2024 Jul 2;96(26):10860-10869. doi: 10.1021/acs.analchem.4c02445. Epub 2024 Jun 18.

Abstract

Single-molecule localization microscopy (SMLM) requires high-intensity laser irradiation, typically exceeding kW/cm2, to yield a sufficient photon count. However, this intense visible light exposure incurs substantial cellular toxicity, hindering its use in living cells. Here, we developed a class of near-infrared (NIR) spontaneously blinking fluorophores for SMLM. These NIR fluorophores are a combination of rhodamine spirolactams and merocyanine derivatives, where the rhodamine spirolactam component converts between a bright and dark state based on pH-dependent spirocyclization and merocyanine derivatives shift the excitation wavelength into the infrared. Single-molecule characterizations demonstrated their potential for SMLM. At a moderate power density of 3.93 kW/cm2, these probes exhibit duty cycle as low as 0.18% and an emission rate as high as 26,700 photons/s. Phototoxicity assessment under single-molecule imaging conditions reveals that NIR illumination (721 nm) minimizes harm to living cells. Employing these NIR fluorophores, we successfully captured time-lapse super-resolution tracking of mitochondria at a Fourier ring correlation (FRC) resolution of 69.4 nm and reconstructed the ultrastructures of endoplasmic reticulum (ER) in living cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Benzopyrans
  • Cell Survival / drug effects
  • Fluorescent Dyes* / chemistry
  • HeLa Cells
  • Humans
  • Indoles / chemistry
  • Infrared Rays*
  • Microscopy, Fluorescence
  • Mitochondria
  • Rhodamines / chemistry

Substances

  • Fluorescent Dyes
  • Indoles
  • Rhodamines
  • merocyanine
  • Benzopyrans