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Correlative super-resolution microscopy with deep UV reactivation.

Kirti Prakash1,2

  • 1Division of Molecular Pathology, The Institute of Cancer Research, London, UK.

Journal of Microscopy
|December 25, 2023
PubMed
Summary

Correlative super-resolution microscopy combines techniques like STED and SMLM. Using deep ultraviolet wavelengths from a Mercury arc lamp can recover STED photobleaching and reactivate fluorophores for SMLM imaging.

Keywords:
LFSMSMLMSTEDUV reactivationcorrelative microscopysinge‐molecule imagingsuper‐resolution microscopy

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Area of Science:

  • Optical microscopy
  • Biophysics
  • Cell biology

Background:

  • Correlative super-resolution microscopy aims to visualize biological structures beyond the diffraction limit.
  • Combining deterministic stimulated emission depletion (STED) and stochastic single-molecule localization microscopy (SMLM) presents significant challenges.
  • Key challenges include fluorophore photobleaching during STED and the need for subsequent fluorophore reactivation for SMLM.

Purpose of the Study:

  • To investigate a method for overcoming photobleaching limitations in correlative STED and SMLM.
  • To demonstrate the utility of deep ultraviolet (DBUE) wavelengths for fluorophore reactivation.
  • To identify optimal fluorophores and imaging media for combined STED and SMLM.

Main Methods:

  • Utilized deep ultraviolet (DBUE) wavelengths from a Mercury (Hg) arc lamp.
  • Applied DBUE irradiation to recover STED-induced photobleaching.
  • Assessed fluorophore reactivation for subsequent SMLM imaging.

Main Results:

  • Deep ultraviolet (DBUE) wavelengths effectively recovered STED-induced photobleaching.
  • DBUE irradiation successfully reactivated single molecules for SMLM imaging.
  • Alexa Fluor 594 and Prolong Diamond were identified as suitable fluorophores and imaging media for correlative STED and SMLM.

Conclusions:

  • Deep ultraviolet (DBUE) excitation offers a viable solution for fluorophore reactivation in correlative STED and SMLM.
  • This approach facilitates seamless integration of STED and SMLM modalities.
  • The combination of Alexa Fluor 594 and Prolong Diamond supports robust correlative super-resolution imaging.