Exploiting the tunability of stimulated emission depletion microscopy for super-resolution imaging of nuclear structures

Maria J Sarmento, Michele Oneto, Simone Pelicci, Luca Pesce, Lorenzo Scipioni, Mario Faretta, Laura Furia, Gaetano Ivan Dellino, Pier Giuseppe Pelicci, Paolo Bianchini, Alberto Diaspro, Luca Lanzanò

Research output: Contribution to journalArticle

Abstract

Imaging of nuclear structures within intact eukaryotic nuclei is imperative to understand the effect of chromatin folding on genome function. Recent developments of super-resolution fluorescence microscopy techniques combine high specificity, sensitivity, and less-invasive sample preparation procedures with the sub-diffraction spatial resolution required to image chromatin at the nanoscale. Here, we present a method to enhance the spatial resolution of a stimulated-emission depletion (STED) microscope based only on the modulation of the STED intensity during the acquisition of a STED image. This modulation induces spatially encoded variations of the fluorescence emission that can be visualized in the phasor plot and used to improve and quantify the effective spatial resolution of the STED image. We show that the method can be used to remove direct excitation by the STED beam and perform dual color imaging. We apply this method to the visualization of transcription and replication foci within intact nuclei of eukaryotic cells.

Original languageEnglish
Pages (from-to)3415
JournalNature Communications
Volume9
Issue number1
DOIs
Publication statusPublished - Aug 24 2018

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Keywords

  • Cell Nucleus/metabolism
  • Cell Nucleus Structures
  • Humans
  • Microscopy, Fluorescence/methods

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