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

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

doi:10.1038/s41467-018-05963-2

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.

Highlights

Imaging of nuclear structures within intact eukaryotic nuclei is imperative to understand the effect of chromatin folding on genome function
In modulation-enhanced stimulated-emission depletion (STED) microscopy (M-STED), the information required for resolution improvement is encoded within the modulation of the STED power during the acquisition of a STED image
The analysis of these variations can be performed with the same separation of photons by lifetime tuning (SPLIT) algorithm that was applied to time-resolved images acquired on a CW-STED microscope[28]

Summary

Introduction

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. The recent development of the so-called super-resolution fluorescence microscopy (SRM) techniques is filling the gap between these two approaches, by combining high specificity, sensitivity, and less-invasive sample preparation procedures with sub-diffraction spatial resolution (1–100 nm). For this reason, SRM methods are well suited to study chromatin spatial arrangement at the nanoscale within intact nuclei. This unique property has been largely exploited in STED-based spot-variation fluorescence correlation spectroscopy (FCS) for measuring molecular diffusion at different spatial scales[30,31] but, to the best of our knowledge, has not been used to improve super-resolved imaging

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Results

Conclusion