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Related Concept Videos

Super-resolution Fluorescence Microscopy01:37

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Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been...
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Updated: Sep 9, 2025

Conducting Multiple Imaging Modes with One Fluorescence Microscope
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Enhanced Single-Molecule Imaging via Intersystem Crossing Suppression.

Fan Ding1, Song Chen1, Fei Du1

  • 1Department of Chemistry and Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials, Fudan University, Shanghai 200438, China.

Journal of the American Chemical Society
|August 28, 2025
PubMed
Summary
This summary is machine-generated.

Researchers developed a new Cy5 dye derivative, Cy5-N, that significantly enhances photostability and signal-to-noise ratio for single-molecule imaging. This innovation overcomes limitations of traditional Cy5 dyes, enabling clearer, longer observations in biological samples.

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Area of Science:

  • Chemical Biology
  • Spectroscopy
  • Biophysics

Background:

  • Cyanine dye 5 (Cy5) is crucial for single-molecule imaging but suffers from photobleaching and signal intermittency due to triplet-state formation.
  • The triplet state of Cy5 promotes reactive oxygen species (ROS) generation and is vulnerable to nucleophilic attacks, limiting its use in biological systems.

Purpose of the Study:

  • To engineer novel phenyl-substituted Cy5 derivatives that suppress intersystem crossing (ISC) and reduce triplet-state population.
  • To enhance the photostability, reduce intermittency, and improve the signal-to-noise ratio (SNR) of Cy5 for advanced bioimaging applications.

Main Methods:

  • Computational chemistry for theoretical calculations.
  • Transient absorption spectroscopy to validate ISC suppression.
  • Synthesis and characterization of phenyl-substituted Cy5 derivatives, including Cy5-N.
  • Single-molecule imaging experiments in vitro and in live U2OS cells.

Main Results:

  • Engineered Cy5 derivatives demonstrated suppressed ISC.
  • The lead compound, Cy5-N, showed a 790% increase in photostability and a 2.3-fold higher SNR compared to standard Cy5.
  • Cy5-N exhibited continuous emission and resistance to nucleophilic attack (e.g., from β-mercaptoethanol).
  • Successful long-term single-molecule tracking of membrane proteins in live cells using Cy5-N with improved SNR.

Conclusions:

  • A novel molecular design strategy effectively enhances cyanine dye performance for single-molecule imaging.
  • Cy5-N offers superior photostability and SNR, overcoming key limitations of conventional Cy5.
  • This advancement has significant potential for improving bioimaging and sensing technologies.