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Graphene- and metal-induced energy transfer for single-molecule imaging and live-cell nanoscopy with (sub)-nanometer axial resolution

Nature Protocols volume 16, pages 3695–3715 (2021)Cite this article

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Abstract

Super-resolution fluorescence imaging that surpasses the classical optical resolution limit is widely utilized for resolving the spatial organization of biological structures at molecular length scales. In one example, single-molecule localization microscopy, the lateral positions of single molecules can be determined more precisely than the diffraction limit if the camera collects individual photons separately. Using several schemes that introduce engineered optical aberrations in the imaging optics, super-resolution along the optical axis (perpendicular to the sample plane) has been achieved, and single-molecule localization microscopy has been successfully applied for the study of 3D biological structures. Nonetheless, the achievable axial localization accuracy is typically three to five times worse than the lateral localization accuracy. Only a few exceptional methods based on interferometry exist that reach nanometer 3D super-resolution, but they involve enormous technical complexity and restricted sample preparations that inhibit their widespread application. We developed metal-induced energy transfer imaging for localizing fluorophores along the axial direction with nanometer accuracy, using only a conventional fluorescence lifetime imaging microscope. In metal-induced energy transfer, experimentally measured fluorescence lifetime values increase linearly with axial distance in the range of 0–100 nm, making it possible to calculate their axial position using a theoretical model. If graphene is used instead of the metal (graphene-induced energy transfer), the same range of lifetime values occurs over a shorter axial distance (~25 nm), meaning that it is possible to get very accurate axial information at the scale of a membrane bilayer or a molecular complex in a membrane. Here, we provide a step-by-step protocol for metal- and graphene-induced energy transfer imaging in single molecules, supported lipid bilayer and live-cell membranes. Depending on the sample preparation time, the complete duration of the protocol is 1–3 d.

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Fig. 1: Working principle of MIET and GIET.
Fig. 2: Experimental setup and work design.
Fig. 3: Orientation of fluorescent emitters.
Fig. 4: Single-molecule localization using GIET.
Fig. 5: GIET experiments on SLB.
Fig. 6: Comparison of basal membrane profiles of three different cell types.
Fig. 7: MIET GUI.

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Data availability

The raw data files corresponding to Figs. 3–6 can be downloaded from https://data.goettingen-research-online.de/dataset.xhtml?persistentId=doi:10.25625/NIDERQ.

Code availability

A custom-written open-source MATLAB-based MIET GUI is available from https://projects.gwdg.de/projects/miet/repository/raw/MIET_GUI.zip?rev=ZIP. The MATLAB-based software package for fluorescence lifetime fitting is available free of charge at https://www.joerg-enderlein.de/software.

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Acknowledgements

N.K. and A.G. are grateful to the Deutsche Forschungsgemeinschaft (DFG) for funding via project A06 of the Collaborative Research Centre SFB 860. J.E. acknowledges funding by the DFG under Germany’s Excellence Strategy—EXC 2067/1- 390729940, and financing by the European Research Council (ERC) via project ‘smMIET’ (grant agreement 884488) under the European Union’s Horizon 2020 research and innovation program.

Author information

Authors and Affiliations

  1. Third Institute of Physics–Biophysics, Georg August University, Göttingen, Germany

    Arindam Ghosh, Alexey I. Chizhik & Jörg Enderlein

  2. Rosalind Franklin Institute, Didcot, UK

    Narain Karedla

  3. Kennedy Institute for Rheumatology, University of Oxford, Oxford, UK

    Narain Karedla

  4. Cluster of Excellence ‘Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells’ (MBExC), Georg August University, Göttingen, Germany

    Jörg Enderlein

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  1. Arindam Ghosh
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Contributions

J.E. conceived the project and helped with data analysis; A.G., A.I.C. and N.K. performed experiments and analyzed the data; A.G. and J.E. wrote the manuscript with the input of all other authors.

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Correspondence to Jörg Enderlein.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Yuval Garini and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Chizhik, A. I. et al. Nat. Photonics 8, 124–127 (2014): https://doi.org/10.1038/nphoton.2013.345

Karedla, N. et al. ChemPhysChem 15, 705–711 (2014): https://doi.org/10.1002/cphc.201300760

Ghosh, A. et al. Nat. Photonics 13, 860–865 (2019): https://doi.org/10.1038/s41566-019-0510-7

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Supplementary Notes 1–4 and Supplementary Fig. 1.

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Ghosh, A., Chizhik, A.I., Karedla, N. et al. Graphene- and metal-induced energy transfer for single-molecule imaging and live-cell nanoscopy with (sub)-nanometer axial resolution. Nat Protoc 16, 3695–3715 (2021). https://doi.org/10.1038/s41596-021-00558-6

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