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Background reduction in STED-FCS using coherent-hybrid STED

Aurélien Barbotin, Iztok Urbančič, Silvia Galiani, Christian Eggeling, Martin Booth
doi: https://doi.org/10.1101/2020.01.06.895243
Aurélien Barbotin
1Department of Engineering Science, University of Oxford, Parks Road, Oxford OX1 3PJ, UK
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Iztok Urbančič
2MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK
3“Jožef Stefan” Institute, Jamova cesta 39, SI-1000 Ljubljana, Slovenia
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  • For correspondence: iztok.urbancic@ijs.si
Silvia Galiani
2MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK
4Wolfson Imaging Centre Oxford, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS
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Christian Eggeling
2MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK
4Wolfson Imaging Centre Oxford, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS
5Institute of Applied Optics and Biophysics, Friedrich-Schiller-University Jena, Max-Wien Platz 4, 07743 Jena, Germany
6Leibniz Institute of Photonic Technology e.V., Albert-Einstein-Strasse 9, 07745 Jena, Germany
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Martin Booth
1Department of Engineering Science, University of Oxford, Parks Road, Oxford OX1 3PJ, UK
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Abstract

Fluorescence correlation spectroscopy (FCS) is a valuable tool to study the molecular dynamics of living cells. When used together with a super-resolution stimulated emission depletion (STED) microscope, STED-FCS can measure diffusion processes at the nanoscale in living cells. In twodimensional (2D) systems like the cellular plasma membrane, a ring-shaped depletion focus is most commonly used to increase the lateral resolution, leading to more than 25-fold decrease in the observation volumee, reaching the relevant scale of supramolecular arrangements. However, STED-FCS faces severe limitations when measuring diffusion in three dimensions (3D), largely due to the spurious background contributions from undepleted areas of the excitation focus that reduce the signal quality and ultimately limit the resolution. In this paper, we investigate how different STED confinement modes can mitigate this issue. By simulations as well as experiments with fluorescent probes in solution and in cells, we demonstrate that the coherent-hybrid (CH) depletion pattern reduces background most efficiently and thus provides superior signal quality under comparable reduction of the observation volume. Featuring also the highest robustness to common optical aberrations, CH-STED can be considered the method of choice for reliable STED-FCS based investigations of 3D diffusion on the sub-diffraction scale.

Footnotes

  • ↵* christian.eggeling{at}rdm.ox.ac.uk

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted January 06, 2020.
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Background reduction in STED-FCS using coherent-hybrid STED
Aurélien Barbotin, Iztok Urbančič, Silvia Galiani, Christian Eggeling, Martin Booth
bioRxiv 2020.01.06.895243; doi: https://doi.org/10.1101/2020.01.06.895243
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Background reduction in STED-FCS using coherent-hybrid STED
Aurélien Barbotin, Iztok Urbančič, Silvia Galiani, Christian Eggeling, Martin Booth
bioRxiv 2020.01.06.895243; doi: https://doi.org/10.1101/2020.01.06.895243

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