Abstract
In the mammalian brain, a large network of excitable and modulatory cells efficiently processes, analyzes and stores vast amounts of information. The brain’s anatomy influences the flow of neural information between neurons and glia, from which all thought, emotion and action arises. Consequently, one of the grand challenges in neuroscience is to uncover the finest structural details of the brain in the context of its overall architecture. Recent developments in microscopy and biosensors have enabled the investigation of brain microstructure and function with unprecedented specificity and resolution, dendritic spines being an exemplary case, which has provided deep insights into neuronal mechanisms of higher brain function, such as learning and memory. As diffraction-limited light microscopy methods cannot resolve the fine details of brain cells (the ‘anatomical ground truth’), electron microscopy is used instead to contextualize functional signals. This approach can be quite unsatisfying given the fragility and dynamic nature of the structures under investigation. We have recently developed a method for combining super-resolution stimulated emission depletion microscopy with functional measurements in brain slices, offering nanoscale resolution in functioning brain structures. We describe how to concurrently perform morphological and functional imaging with a confocal STED microscope. Specifically, the procedure guides the user on how to record astrocytic Ca2+ signals at tripartite synapses, outlining a framework for analyzing structure–function relationships of brain cells at nanoscale resolution. The imaging requires 2–3 h and the image analysis between 2 h and 2 d.
Key points
The procedure covers Ca2+ imaging at nanoscale resolution of synapses and explains how to correlate functional Ca2+ activity with morphological data in neurons, astrocytes and the extracellular space.
Correlative live STED offers an alternative to correlative light and electron microscopy to study the physiological function and nanoscale morphology of cells and organelles.
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Fig. 1: STED principle and setup alignment.
Fig. 2: Outline of correlative live-cell STED microscopy.
Fig. 3: Microinjection of brain slices.
Fig. 4: Mounting the slice in the chamber.
Fig. 5: Live STED imaging of brain slices.
Fig. 6: Mapping the functional readout on super-resolved image for the correlative live STED microscopy.
Fig. 7: Analysis of SUSHI images.
Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request. Further information and requests for resources and reagents should be directed to and will be provided by the lead contact, U.V. Nägerl (valentin.nagerl@med.uni-goettingen.de).
References
Arizono, M., Idziak, A., Quici, F. & Nagerl, U. V. Getting sharper: the brain under the spotlight of super-resolution microscopy. Trends Cell Biol. 33, 148–161 (2023).
ArticleCASPubMedGoogle Scholar
Balzarotti, F. et al. Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science 355, 606–612 (2017).
ArticleCASPubMedGoogle Scholar
Wirth, J. O. et al. MINFLUX dissects the unimpeded walking of kinesin-1. Science 379, 1004–1010 (2023).
ArticlePubMedGoogle Scholar
Boden, A. et al. Volumetric live cell imaging with three-dimensional parallelized RESOLFT microscopy. Nat. Biotechnol. 39, 609–618 (2021).
ArticleCASPubMedGoogle Scholar
Calovi, S., Soria, F. N. & Tonnesen, J. Super-resolution STED microscopy in live brain tissue. Neurobiol. Dis. 156, 105420 (2021).
ArticleCASPubMedGoogle Scholar
Fuhrmann, M. et al. Super-resolution microscopy opens new doors to life at the nanoscale. J. Neurosci. 42, 8488–8497 (2022).
ArticleCASPubMedPubMed CentralGoogle Scholar
Nagerl, U. V., Willig, K. I., Hein, B., Hell, S. W. & Bonhoeffer, T. Live-cell imaging of dendritic spines by STED microscopy. Proc. Natl Acad. Sci. USA 105, 18982–18987 (2008).
ArticleCASPubMedPubMed CentralGoogle Scholar
Tonnesen, J., Nadrigny, F., Willig, K. I., Wedlich-Soldner, R. & Nagerl, U. V. Two-color STED microscopy of living synapses using a single laser-beam pair. Biophys. J. 101, 2545–2552 (2011).
ArticleCASPubMedPubMed CentralGoogle Scholar
Urban, N. T., Willig, K. I., Hell, S. W. & Nagerl, U. V. STED nanoscopy of actin dynamics in synapses deep inside living brain slices. Biophys. J. 101, 1277–1284 (2011).
ArticleCASPubMedPubMed CentralGoogle Scholar
Riedl, J. et al. Lifeact: a versatile marker to visualize F-actin. Nat. Methods 5, 605–607 (2008).
ArticleCASPubMedPubMed CentralGoogle Scholar
Tonnesen, J., Katona, G., Rozsa, B. & Nagerl, U. V. Spine neck plasticity regulates compartmentalization of synapses. Nat. Neurosci. 17, 678–685 (2014).
ArticleCASPubMedGoogle Scholar
Chereau, R., Saraceno, G. E., Angibaud, J., Cattaert, D. & Nagerl, U. V. Superresolution imaging reveals activity-dependent plasticity of axon morphology linked to changes in action potential conduction velocity. Proc. Natl Acad. Sci. USA 114, 1401–1406 (2017).
ArticleCASPubMedPubMed CentralGoogle Scholar
Inavalli, V. et al. A super-resolution platform for correlative live single-molecule imaging and STED microscopy. Nat. Methods 16, 1263–1268 (2019).
ArticleCASPubMedGoogle Scholar
Bethge, P., Chereau, R., Avignone, E., Marsicano, G. & Nagerl, U. V. Two-photon excitation STED microscopy in two colors in acute brain slices. Biophys. J. 104, 778–785 (2013).
ArticleCASPubMedPubMed CentralGoogle Scholar
Pfeiffer, T. et al. Chronic 2P-STED imaging reveals high turnover of dendritic spines in the hippocampus in vivo. eLife 7, e34700 (2018).
ArticlePubMedPubMed CentralGoogle Scholar
Bancelin, S., Mercier, L., Murana, E. & Nagerl, U. V. Aberration correction in stimulated emission depletion microscopy to increase imaging depth in living brain tissue. Neurophotonics 8, 035001 (2021).
ArticleCASPubMedPubMed CentralGoogle Scholar
Le Bourdelles, G., Mercier, L., Roos, J., Bancelin, S. & Nagerl, U. V. Impact of a tilted coverslip on two-photon and STED microscopy. Biomed. Opt. Express 15, 743–752 (2024).
ArticlePubMedPubMed CentralGoogle Scholar
Bancelin, S. et al. Imaging dendritic spines in the hippocampus of a living mouse by 3D-stimulated emission depletion microscopy. Neurophotonics 10, 044402 (2023).
ArticlePubMedPubMed CentralGoogle Scholar
Panatier, A., Arizono, M. & Nagerl, U. V. Dissecting tripartite synapses with STED microscopy. Philos. Trans. R. Soc. Lond. B 369, 20130597 (2014).
ArticleGoogle Scholar
Refaeli, R. et al. Features of hippocampal astrocytic domains and their spatial relation to excitatory and inhibitory neurons. Glia 69, 2378–2390 (2021).
ArticleCASPubMedPubMed CentralGoogle Scholar
Salmon, C. K. et al. Organizing principles of astrocytic nanoarchitecture in the mouse cerebral cortex. Curr. Biol. 33, 957–972 e955 (2023).
ArticleCASPubMedGoogle Scholar
Arizono, M. & Nagerl, U. V. Deciphering the functional nano-anatomy of the tripartite synapse using stimulated emission depletion microscopy. Glia 70, 607–618 (2022).
ArticlePubMedGoogle Scholar
Arizono, M. et al. Structural basis of astrocytic Ca2+ signals at tripartite synapses. Nat. Commun. 11, 1906 (2020).
ArticleCASPubMedPubMed CentralGoogle Scholar
Tonnesen, J., Inavalli, V. & Nagerl, U. V. Super-resolution imaging of the extracellular space in living brain tissue. Cell 172, 1108–1121 e1115 (2018).
ArticlePubMedGoogle Scholar
Nicholson, C. & Hrabetova, S. Brain extracellular space: the final frontier of neuroscience. Biophys. J. 113, 2133–2142 (2017).
ArticleCASPubMedPubMed CentralGoogle Scholar
Tonnesen, J., Hrabetova, S. & Soria, F. N. Local diffusion in the extracellular space of the brain. Neurobiol. Dis. 177, 105981 (2023).
ArticlePubMedGoogle Scholar
Nicholson, C., Chen, K. C., Hrabetova, S. & Tao, L. Diffusion of molecules in brain extracellular space: theory and experiment. Prog. Brain Res. 125, 129–154 (2000).
ArticleCASPubMedGoogle Scholar
Lamprecht, R. & LeDoux, J. Structural plasticity and memory. Nat. Rev. Neurosci. 5, 45–54 (2004).
ArticleCASPubMedGoogle Scholar
Zoghbi, H. Y. & Bear, M. F. Synaptic dysfunction in neurodevelopmental disorders associated with autism and intellectual disabilities. Cold Spring Harb. Perspect. Biol. 4, a009886 (2012).
ArticlePubMedPubMed CentralGoogle Scholar
Padmanabhan, P., Kneynsberg, A. & Gotz, J. Super-resolution microscopy: a closer look at synaptic dysfunction in Alzheimer disease. Nat. Rev. Neurosci. 22, 723–740 (2021).
ArticleCASPubMedGoogle Scholar
Valiente-Gabioud, A. A. et al. Fluorescent sensors for imaging of interstitial calcium. Nat. Commun. 14, 6220 (2023).
ArticleCASPubMedPubMed CentralGoogle Scholar
Stolp, B. et al. Salivary gland macrophages and tissue-resident CD8+ T cells cooperate for homeostatic organ surveillance. Sci. Immunol. 5, eaaz4371 (2020).
ArticleCASPubMedGoogle Scholar
Dembitskaya, Y. et al. Shadow imaging for panoptical visualization of brain tissue in vivo. Nat. Commun. 14, 6411 (2023).
ArticleCASPubMedPubMed CentralGoogle Scholar
Velicky, P. et al. Dense 4D nanoscale reconstruction of living brain tissue. Nat. Methods 20, 1256–1265 (2023).
ArticleCASPubMedPubMed CentralGoogle Scholar
Korogod, N., Petersen, C. C. & Knott, G. W. Ultrastructural analysis of adult mouse neocortex comparing aldehyde perfusion with cryo fixation. eLife 4, e05793 (2015).
ArticlePubMedPubMed CentralGoogle Scholar
Aggarwal, A. et al. Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission. Nat. Methods 20, 925–934 (2023).
ArticleCASPubMedPubMed CentralGoogle Scholar
Shen, Y., Nasu, Y., Shkolnikov, I., Kim, A. & Campbell, R. E. Engineering genetically encoded fluorescent indicators for imaging of neuronal activity: progress and prospects. Neurosci. Res. 152, 3–14 (2020).
ArticlePubMedGoogle Scholar
Jeong, S., Widengren, J. & Lee, J. C. Fluorescent probes for STED optical nanoscopy. Nanomaterials 12, 21 (2021).
ArticlePubMedPubMed CentralGoogle Scholar
Grieger, J. C., Choi, V. W. & Samulski, R. J. Production and characterization of adeno-associated viral vectors. Nat. Protoc. 1, 1412–1428 (2006).
ArticleCASPubMedGoogle Scholar
O’Brien, J. A. & Lummis, S. C. Biolistic transfection of neuronal cultures using a hand-held gene gun. Nat. Protoc. 1, 977–981 (2006).
ArticlePubMedPubMed CentralGoogle Scholar
Judkewitz, B., Rizzi, M., Kitamura, K. & Hausser, M. Targeted single-cell electroporation of mammalian neurons in vivo. Nat. Protoc. 4, 862–869 (2009).
ArticleCASPubMedGoogle Scholar
Haustein, M. D. et al. Conditions and constraints for astrocyte calcium signaling in the hippocampal mossy fiber pathway. Neuron 82, 413–429 (2014).
ArticleCASPubMedPubMed CentralGoogle Scholar
Gahwiler, B. H. Organotypic monolayer cultures of nervous tissue. J. Neurosci. Methods 4, 329–342 (1981).
ArticleCASPubMedGoogle Scholar
Stoppini, L., Buchs, P. A. & Muller, D. A simple method for organotypic cultures of nervous tissue. J. Neurosci. Methods 37, 173–182 (1991).
ArticleCASPubMedGoogle Scholar
Levet, F., Tonnesen, J., Nagerl, U. V. & Sibarita, J. B. SpineJ: a software tool for quantitative analysis of nanoscale spine morphology. Methods 174, 49–55 (2020).
ArticleCASPubMedGoogle Scholar
Kimura, T. et al. Production of adeno-associated virus vectors for in vitro and in vivo applications. Sci. Rep. 9, 13601 (2019).
ArticlePubMedPubMed CentralGoogle Scholar
Stobart, J. L. et al. Cortical circuit activity evokes rapid astrocyte calcium signals on a similar timescale to neurons. Neuron 98, 726–735 e724 (2018).
ArticleCASPubMedGoogle Scholar
Feng, G. et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41–51 (2000).
ArticleCASPubMedGoogle Scholar
Cheers, M. S. & Ettensohn, C. A. Rapid microinjection of fertilized eggs. Methods Cell Biol. 74, 287–310 (2004).
ArticlePubMedGoogle Scholar
Francis, M. et al. Automated region of interest analysis of dynamic Ca2+ signals in image sequences. Am. J. Physiol. Cell Physiol. 303, C236–C243 (2012).
ArticleCASPubMedPubMed CentralGoogle Scholar
Henneberger, C. et al. LTP induction boosts glutamate spillover by driving withdrawal of perisynaptic astroglia. Neuron 108, 919–936 e911 (2020).
ArticleCASPubMedPubMed CentralGoogle Scholar
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Acknowledgements
This work was supported by the Agence Nationale de la Recherche (ANR-18-CE16-0018), Fondation pour la Recherche Médicale (FRM), Human Frontier Science Program (RGP0041/2019), European Research Council (Synergy grant ENSEMBLE, 951294) to U.V.N. M.A. was supported by Brain Science Foundation, JSPS (grant number 22568099) and JST FOREST Program (grant number JPMJFR2141), and A.I. was supported by a PhD fellowship from the Bordeaux Neurocampus Graduate Program. We thank M. Sherwood and members of the Nägerl team for comments on the manuscript.
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Authors and Affiliations
Interdisciplinary Institute for Neuroscience, University of Bordeaux, CNRS UMR 5297, Bordeaux, France
Misa Arizono, Agata Idziak & U. Valentin Nägerl
The Hakubi Center for Advanced Research, Kyoto University, Kyoto, Japan
Misa Arizono
Department of Pharmacology, Kyoto University Graduate School of Medicine, Kyoto University, Kyoto, Japan
Misa Arizono & Agata Idziak
Department of Anatomy and Cell Biology, University Medical Center, Georg-August-University of Göttingen, Göttingen, Germany
U. Valentin Nägerl
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Misa Arizono
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V.N. and M.A. designed the original protocol. All authors refined and improved the protocol and wrote the manuscript.
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Correspondence to Misa Arizono or U. Valentin Nägerl.
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Key references
Arizono, M. et al. Nat. Commun. 11, 1906 (2020): https://doi.org/10.1038/s41467-020-15648-4
Tønnesen, J. et al. Cell 172, 1108–1121 (2018): https://doi.org/10.1016/j.cell.2018.02.007
Dembitskaya, Y. et al. Nat. Commun. 14, 6411 (2023): https://doi.org/10.1038/s41467-023-42055-2
Arizono M, et al. Glia. 69, 1605–1613 (2021): https://doi.org/10.1002/glia.23995
Tønnesen, J. et al. Nat. Neurosci. 17, 678–685 (2014): https://doi.org/10.1038/nn.3682
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Arizono, M., Idziak, A. & Nägerl, U.V. Live STED imaging of functional neuroanatomy. Nat Protoc (2025). https://doi.org/10.1038/s41596-024-01132-6
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Received:17 May 2024
Accepted:11 December 2024
Published:14 March 2025
DOI:https://doi.org/10.1038/s41596-024-01132-6
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