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Stimulated emission depletion microscopy

Nature Reviews Methods Primers volume 4, Article number: 56 (2024) Cite this article

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Abstract

In this Primer, we focus on the most recent advancements in stimulated emission depletion (STED) microscopy, encompassing optics, computational microscopy and probes design, which enable STED imaging to open new observation windows in challenging samples such as living cells and tissues. We showcase applications in which STED data have been essential to gain new biological insights in various cell types and model systems. Finally, we discuss what standardization will be important in our view to further advance STED imaging, including open and shareable software, analysis pipelines, data repositories and sample preparation protocols.

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Fig. 1: Principles of stimulated emission depletion microscopy.
Fig. 2: Typical stimulated emission depletion setup, depletion patterns and beam shaping using a spatial light modulator.
Fig. 3: Time-resolved stimulated emission depletion microscopy.
Fig. 4: Stimulated emission depletion-fluorescence correlation spectroscopy and stimulated emission depletion-image-scanning microscopy.
Fig. 5: Fluorophore types and labelling methods used for stimulated emission depletion microscopy.
Fig. 6: Examples of 2D and 3D stimulated emission depletion imaging data.
Fig. 7: Novel biological insights using stimulated emission depletion microscopy.
Fig. 8: Deep learning for stimulated emission depletion microscopy.

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Acknowledgements

K.Y.H. thanks the NIH (R35GM138039) for supporting this work. I.T. thanks the ERC-CoG (101002490 — InSpIRe) for supporting this work. F.B. acknowledges the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) — Project Number 278001972 — TRR 186 grant and a HFSP Early career award that supports C.R.-R. J.A. thanks the Swedish Research Council (2022-06139_VR) for supporting this work. G.V. thanks all members of the Molecular Microscopy and Spectroscopy laboratory at the Istituto Italiano di Tecnologia for their continuous discussions during the writing process and the European Research Council for their support of his research through the BrightEyes Consolidator grant (No. 818699). G.L., R.G. and V.T.N. thank the Max Planck Society for supporting this work. V.T.N. is supported by the PhD program Genome Science — International Max Planck Research School.

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Authors and Affiliations

  1. Chromatin Labeling and Imaging Group, Department of NanoBiophotonics, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany

    Gražvydas Lukinavičius, Rūta Gerasimaitė & Văn Thắng Nguyễn

  2. Department of Biophysical Imaging, Leibniz Institute of Photonic Technology, Jena, Germany

    Jonatan Alvelid

  3. Department of Applied Physics and SciLifeLab, KTH Royal Institute of Technology, Stockholm, Sweden

    Jonatan Alvelid & Ilaria Testa

  4. Institut für Biochemie, Freie Universität Berlin, Berlin, Germany

    Carmen Rodilla-Ramirez & Francesca Bottanelli

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

    Giuseppe Vicidomini

  6. CREOL, The College of Optics and Photonics, University of Central Florida, Orlando, FL, USA

    Kyu Young Han

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The authors contributed equally to all aspects of the article.

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Correspondence to Ilaria Testa.

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G.V. has a personal financial interest (co-founder) in Genoa Instruments, a company commercializing laser-scanning microscopy based on single-photon avalanche diode array detectors. The other authors declare no competing interests.

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Supplementary information

Glossary

Cross-section

The probability of particle–photon interaction. A typical absorption cross-section for a fluorophore at room temperature is on the order of 10–16 cm2.

Deformable mirrors

Optical device typically used to shape the wavefront of reflected light.

Depletion

Induced de-excitation of a molecule to the ground state without detectable fluorescence emission.

Fluorescence photo-switching

Property of turning ‘on’ and ‘off’ the fluorescence emission of a molecule with light. Light-induced molecular pathways leading to photo-switching are stimulated emission or other molecular conformational changes connected to spectral shifts.

Fluorogenicity

Ability of the probe to increase fluorescence upon binding to the target. When free in solution, such a probe is only weakly fluorescent. Using fluorogenic probes helps in getting higher contrast images.

Fluorogens

Non-fluorescent small molecule that can become fluorescent after it is complexed with a specific fluorogen-activating protein.

Gated STED

Stimulated emission depletion (STED) technique in which not all detected emitted photons are considered but only the ones delayed with 0.5 ns or more, typically, when compared with the excitation pulse. It is used to filter the signal from unwanted low-resolution photon information emitted before the stimulated emission has taken full effect.

Phasor-based approach

Data visualization approach used in lifetime imaging, which helps to separate molecular species based on the temporal and spectral properties of the fluorescence emission.

Self-labelling protein tag

An enzyme that can be inactivated by suicide inhibition — a substrate analogue that forms an irreversible complex through a covalent bond during the catalysis reaction. The suicide inhibitor can carry any reporter group that generates a detectable signal, that is, fluorophore, radioactive atom, spin label or other.

Separation of photon by lifetime tuning (SPLIT)-STED microscopy

A stimulated emission depletion (STED) microscopy technique that utilizes fluorescence dynamics to select photons containing high spatial resolution information through a computational approach based on the phasor analysis of the fluorescence lifetime emission.

Spatial light modulator

Optical device typically used to modify the phase of reflected or transmitted light.

Spirolactone

A form of a rhodamine featuring a cyclic ester attached to the three-ring xanthene system. Usually, spirolactone formation results in neutralization of positive and negative charges on the fluorophore molecule.

Spot-variation (or diffusion law) FCS

Fluorescence correlation technique capable of unveiling constraints in the dynamics of the investigated molecule or particle. By plotting the apparent diffusion coefficients of the molecule as a function of the observation volume size during fluorescence correlation spectroscopy (FCS) measurements, it is possible to reconstruct the diffusion law, allowing for an understanding of whether the molecule is diffusing freely or is subject to partition dynamics, hopping or other types of mobility.

Zwitterionic state

A state of a rhodamine that is formed after spirolactone cleavage, resulting in the appearance of positive and negative charges on the same dye molecule.

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Lukinavičius, G., Alvelid, J., Gerasimaitė, R. et al. Stimulated emission depletion microscopy. Nat Rev Methods Primers 4, 56 (2024). https://doi.org/10.1038/s43586-024-00335-1

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