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 () and Luca Lanzanò ()
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Maria J. Sarmento: Istituto Italiano di Tecnologia
Michele Oneto: Istituto Italiano di Tecnologia
Simone Pelicci: Istituto Italiano di Tecnologia
Luca Pesce: Istituto Italiano di Tecnologia
Lorenzo Scipioni: Istituto Italiano di Tecnologia
Mario Faretta: European Institute of Oncology
Laura Furia: European Institute of Oncology
Gaetano Ivan Dellino: European Institute of Oncology
Pier Giuseppe Pelicci: European Institute of Oncology
Paolo Bianchini: Istituto Italiano di Tecnologia
Alberto Diaspro: Istituto Italiano di Tecnologia
Luca Lanzanò: Istituto Italiano di Tecnologia

Nature Communications, 2018, vol. 9, issue 1, 1-11

Abstract: 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.

Date: 2018
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DOI: 10.1038/s41467-018-05963-2

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