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STED super-resolved microscopy

Nature Methods volume 15, pages 173–182 (2018)Cite this article

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Abstract

Stimulated emission depletion (STED) microscopy provides subdiffraction resolution while preserving useful aspects of fluorescence microscopy, such as optical sectioning, and molecular specificity and sensitivity. However, sophisticated microscopy architectures and high illumination intensities have limited STED microscopy's widespread use in the past. Here we summarize the progress that is mitigating these problems and giving substantial momentum to STED microscopy applications. We discuss the future of this method in regard to spatiotemporal limits, live-cell imaging and combination with spectroscopy. Advances in these areas may elevate STED microscopy to a standard method for imaging in the life sciences.

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Figure 1: STED microscopy principles.
Figure 2: STED microscopy architectures.
Figure 3: RESCue- and protected-STED microscopy.
Figure 4: Comparison of confocal and STED microscopy images.

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Acknowledgements

We equally acknowledge S. Koho, M. Castello and G. Tortarolo (Istituto Italiano di Tecnologia) for fruitful discussions.

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Authors and Affiliations

  1. Molecular Microscopy and Spectroscopy, Istituto Italiano di Tecnologia, Genoa, Italy

    Giuseppe Vicidomini

  2. Nanoscopy and NIC@IIT, Istituto Italiano di Tecnologia, Genoa, Italy

    Paolo Bianchini & Alberto Diaspro

  3. Department of Physics, University of Genoa, Genoa, Italy

    Alberto Diaspro

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  1. Giuseppe Vicidomini
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Corresponding author

Correspondence to Giuseppe Vicidomini.

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Integrated supplementary information

Supplementary Figure 1 Milestones of the laser technology developments on STED microscopy.1–23

The colour of the milestone year denotes if the associated STED implementation is: (i) obsolete (black); (ii) commercial available (green); routinely used but custom-made (red). The dismissed and current commercial STED microscopes are: Leica TCS-STED (LeTCS, dismissed); Leica TCS-STED-CW (LeTCSCW, dismissed); Leica TCS-STED-3X (LeTCS3X); Abberior STED QUAD Scan (AbQS); Abberior STEDYCON (AbSYC); PicoQuant MicroTime 200 STED (PqMT200); ISS Alba-STED (Alba)

Supplementary Figure 2 Multi-colour STED microscopy.15,21,22,24–47

For each dyes combination, the STED implementation used is reported: custom made (CM) or commercial. Compatible commercial implementations have been also reported. AbQS@775 and AbQS@595 indicated the STED beam's wavelength configuration (775 nm or 595 nm, respectively) for the AbQS commercial systems.

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Supplementary Figures 1–2 and Supplementary Notes 1–4 (PDF 1664 kb)

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Vicidomini, G., Bianchini, P. & Diaspro, A. STED super-resolved microscopy. Nat Methods 15, 173–182 (2018). https://doi.org/10.1038/nmeth.4593

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