Progress in microscopy technology has a long history of triggering major advances in neuroscience. Super-resolution microscopy (SRM), famous for shattering the diffraction barrier of light microscopy, is no exception. SRM gives access to anatomical designs and dynamics of nanostructures, which are impossible to resolve using conventional light microscopy, from the elaborate anatomy of neurons and glial cells, to the organelles and molecules inside of them. In this review, we will mainly focus on a particular SRM technique (STED microscopy), and explain a series of technical developments we have made over the years to make it practical and viable in the field of neuroscience. We will also highlight several neurobiological findings on the dynamic structure-function relationship of neurons and glia cells, which illustrate the value of live-cell STED microscopy, especially when combined with other modern approaches to investigate the nanoscale behavior of brain cells.
Fortschritte in der Mikroskopie-Technik haben in der Vergangenheit immer wieder große Durchbrüche in den Neurowissenschaften ausgelöst. Die superauflösende Fluoreszenzmikroskopie, berühmt für die Durchbrechung der Beugungsgrenze der Lichtmikroskopie, bildet hier keine Ausnahme. Sie ermöglicht beispiellosen Zugang zum anatomischen Aufbau und der Dynamik von Nanostrukturen, die mit konventioneller Lichtmikroskopie nicht auflösbar sind, von der ausgefeilten Anatomie der Nerven- und Gliazellen bis hin zu den Organellen und Proteinen in ihrem Inneren. In diesem Überblicksartikel werden wir hauptsächlich auf die STED-Mikroskopie eingehen und eine Reihe von technischen Neuerungen erläutern, die wir im Laufe der Jahre anwendungsspezifisch dafür entwickelt haben. Wir werden dabei einige unserer neurobiologischen Untersuchungen und Resultate über Synapsen, Gliazellen und den Extrazellulär-Raum vorstellen, wo die ,live-cell‘ STED-Mikroskopie in Kombination mit anderen modernen Ansätzen einen entscheidenden Beitrag leisten konnte.
Funding source: Human Frontier Science Program (HFSP)
Funding source: Agence Nationale de la Recherche (ANR)
Funding source: Japan Society for the Promotion of Science (JSPS)
Funding source: Marie Skłodowska-Curie Program
Funding source: ATIP – Avenir Program (Inserm)
Funding source: EMBO
Funding source: Fondation pour la Recherche Médicale (FRM)
Funding source: Fédération pour la recherche sur le cerveau (FRC)
Funding source: AXA Foundation
Funding source: Lundbeck Foundation
Funding source: Labex BRAIN
Funding source: Boehringer Ingelheim Fonds
About the authors
Dr. Misa Arizono studied neuroscience and received her PhD from the University of Tokyo in 2012 in the Mikoshiba lab. Since 2013, she has been a postdoc in the Nägerl team in Bordeaux. Using cutting-edge microscopy techniques, such as STED and lattice light-sheet calcium imaging, she has been deciphering the biophysical mechanisms that shape the communication between neurons and astrocytes. She has received numerous distinctions, including competitive postdoc fellowships from Japan, the Inoue Research Award for Young Scientists and poster prizes at international workshops and conferences. On weekends, she enjoys cooking while traveling around the world of ideas with podcasts. She loves graphic design and scientific communication. Go explore her website: https://arizono0202.wixsite.com/misa-arizono.
Dr. Stéphane Bancelin studied physics at the École normale supérieure Paris-Saclay and received his PhD in 2013 from the École Polytechnique in Palaiseau, where he worked on SHG microscopy to image connective tissue. During his first postdoc at the University of Quebec in Canada, he developed interferometric approaches in nonlinear microscopy. For his second postdoc, he joined the Nägerl team in Bordeaux, where he is working on the development of super-resolution microscopy to study the cellular mechanisms of memory formation in vivo. He recently obtained a tenured position as CNRS researcher at the Interdisciplinary Institute for Neuroscience.
Dr. Philipp Bethge studied psychology and neurobiology at the University Landau and University of Magdeburg before joining the Nägerl lab for his PhD where he developed 2-photon STED microscopy with pulsed laser sources. He continued as a postdoc with an HFSP fellowship in the lab of Prof. Helmchen at the Univ. of Zurich, working on large field-of-view multiarea multiphoton microscopy. He has become the scientific coordinator of the Helmchen team, but also an avid bird photographer and paragliding dare-devil.
Dr. Ronan Chéreau studied biology at the Ecole Pratique des Hautes Etudes in Paris. He received his PhD from the Univ. of Bordeaux in 2014 under the supervision of Prof. Nägerl. Using STED microscopy, he studied the morpho-functional changes of hippocampal axons during plasticity in mouse brain slices. In 2014 he joined the lab of Prof. Holtmaat in Geneva where he has continued to study the physiology of axons. He is particularly interested in understanding the role of the thalamocortical feedback fibers activity during sensory perception and learning and how this activity is integrated in the cortex.
Agata Idziak studied biomedical science and cellular neuroscience at the Philipps-Universität Marburg in Germany. She pursued her master thesis in the team of Prof. Nägerl, studying perineuronal nets using super-resolution microscopy approaches developed by the team. Winning a highly competitive PhD fellowship from Bordeaux Neurocampus, she lives and learns under the supervision of Misa and Valentin, studying the structure & function of brain extracellular space with an array of cool techniques, including new biosensors, SUSHI, lattice light-sheet microscopy (and soon e-phys). In her free time, she likes to travel, experiment in the kitchen, play tennis and hit the twittersphere.
Dr. V.V.G. Krishna Inavalli obtained his PhD in physics from University of Hyderabad, India, where he developed a method to generate structured light beams with orbital angular momentum and isolated polarization singularities and their applications by demonstrating the rotational Doppler effect. During his first postdoc at the University of Illinois at Urbana Champaign, USA, he developed novel microscopy techniques to image biological tissues and plasmonic nanofilms and worked on techniques for complex wavefront shaping. For his second postdoc, he joined the teams of Valentin Nägerl and Jean-Baptiste Sibarita in Bordeaux, where he developed sharper correlative super-resolution microscopes for cellular neuroscience. Recently, he set sail for the UK, after getting appointed as head of the microscopy group at the Center for Cancer Immunology at the Univ. of Southampton in the UK.
Dr. Thomas Pfeiffer studied molecular biosciences and neuroscience at the University of Heidelberg, Germany. During his master thesis in the group of Prof. Draguhn, he combined calcium imaging and electrophysiological techniques to visualize the activity of hippocampal cell assemblies. Thomas joined the Nägerl lab in Bordeaux for his PhD, studying the dynamics of dendritic spines and microglia using STED and two-photon microscopy in mouse brain slices and in vivo. Thomas is currently a postdoctoral researcher with Prof. Attwell at the University College London in the UK, where he focuses on interactions between brain microvasculature and the immune system. In his next move, he wants to shake up the UK biotech industry.
Dr. Jan Tønnesen studied biology at the University of Copenhagen. He received his PhD from Lund University in Sweden, under the supervision of Prof. Kokaia, where he investigated cell-replacement therapy in Parkinson’s disease and optogenetic control of epileptiform activity. From 2010 to 2016, he was a postdoc and researcher in the lab of Prof. Nägerl, using STED microscopy to study spine plasticity. He won the ‘Great Advances in Biology’ prize from the French Academy of Sciences (2018) for his work on the SUSHI technique. Since 2016, he is a group leader at the Achucarro Basque Center for Neuroscience in Bilbao, where he continues to address questions about synaptic signalling and neuronal excitability using electrophysiological and advanced optical microscopy approaches. When not performing cutting-edge experiments in darkened rooms, he enjoys angling and the outdoors.
Prof. Dr. U. Valentin Nägerl is a professor of neuroscience and bioimaging at the University of Bordeaux, where he leads the research group ‘Synaptic Plasticity and Super-Resolution Microcopy’ and organizes the biophotonics curriculum at the Graduate School for Light Science and Technologies. He studied physics and pre-clinical medicine in Göttingen and got his PhD in neuroscience from UCLA working with Istvan Mody. He did his postdoc with Tobias Bonhoeffer at the Max Planck Institute of Neurobiology, habilitated with Arthur Konnerth at the Technical University of Munich and worked with Stefan W. Hell at the Max Planck Institute for Biophysical Chemistry. In Bordeaux since 2009, he has received several awards and distinctions for his work on the nano-mechanisms of brain function (Equipe Inserm Avenir, 2009; HFSP award, 2010; Equipe FRM award, 2016; Senior membership of the Institut Universitaire de France, 2017; Great Advances in Biology Prize, French Academy of Sciences, 2018; HFSP award, 2020). For the next few years, his research will be supported by an ‘ERC Synergy Grant’ to conduct frontier research on the extracellular space of the brain. Perfecting his skills during the pandemic, he enjoys baking bagels and Kaiserschmarrn for his home team. Web page: https://www.iins.u-bordeaux.fr/NAGERL, Twitter: https://twitter.com/NagerlL.
We would like to warmly thank all the people who have contributed to our efforts over the years, including lab members and visitors (Angeles Almeida, Aude Panatier, Cordelia Imig, Elena Avignone, Emanuele Murana, Fabien Nadrigny, Julie Angibaud, Kosuke Okuda, Lasani Wijetunge, Luc Mercier, Mark Sherwood, Martin Lenz, Motohiro Nozumi, Stefanie Poll, Sun Kwang Kim) and collaborators (Daniel Cattaert, Daniel Choquet, David DiGregorio, Dmitri Rusakov, Giovanni Marsicano, Jean-Baptiste Sibarita, Jerome Badaut, Martin Fuhrmann, Peter C. Kind, Serge Marty, Stefan W. Hell, Stephane Oliet).
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: We are also grateful for the financial support from these funding agencies: Human Frontier Science Program (HFSP), Agence Nationale de la Recherche (ANR), Japan Society for the Promotion of Science (JSPS), Marie Skłodowska-Curie Program, ATIP – Avenir Program (Inserm), EMBO, Fondation pour la Recherche Médicale (FRM), Fédération pour la recherche sur le cerveau (FRC), AXA Foundation, Lundbeck Foundation, Labex BRAIN, Boehringer Ingelheim Fonds.
Conflict of interest statement: The authors declare no conflicts of interest regarding this article.
Adrian, M., Kusters, R., Wierenga, C.J., Storm, C., Hoogenraad, C.C., and Kapitein, L.C. (2014). Barriers in the brain: Resolving dendritic spine morphology and compartmentalization. Front. Neuroanat.8, 142, https://doi.org/10.3389/fnana.2014.00142.Search in Google Scholar PubMed PubMed Central
Alle, H. and Geiger, J.R. (2006). Combined analog and action potential coding in hippocampal mossy fibers. Science311, 1290–1293, https://doi.org/10.1126/science.1119055.Search in Google Scholar PubMed
Angibaud, J., Mascalchi, P., Poujol, C., and Nägerl, U.V. (2020). A simple tissue clearing method for increasing the depth penetration of STED microscopy of fixed brain slices. J. Phys. Appl. Phys.53, 184001, https://doi.org/10.1088/1361-6463/ab6f1b.Search in Google Scholar
Arizono, M., Inavalli, V.V.G.K., and Nägerl, U.V. (2021). Super-resolution shadow imaging reveals local remodeling of astrocytic microstructures and brain extracellular space after osmotic challenge. bioRxiv, https://doi.org/10.1101/2021.01.05.425369.Search in Google Scholar
Arizono, M., Inavalli, V.V.G.K., Panatier, A., Pfeiffer, T., Angibaud, J., Levet, F., Ter Veer, M.J.T., Stobart, J., Bellocchio, L., Mikoshiba, K., et al.. (2020). Structural basis of astrocytic Ca2+ signals at tripartite synapses. Nat. Commun.11, 1906, https://doi.org/10.1038/s41467-020-15648-4.Search in Google Scholar PubMed PubMed Central
Attardo, A., Fitzgerald, J.E., and Schnitzer, M.J. (2015). Impermanence of dendritic spines in live adult CA1 hippocampus. Nature523, 592–596, https://doi.org/10.1038/nature14467.Search in Google Scholar PubMed PubMed Central
Balzarotti, F., Eilers, Y., Gwosch, K.C., Gynnå, A.H., Westphal, V., Stefani, F.D., Elf, J., and Hell, S.W. (2017). Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science355, 606–612, https://doi.org/10.1126/science.aak9913.Search in Google Scholar PubMed
Bancelin, S., Mercier, L., Murana, E., and Nägerl, V. (2021). Aberration correction in STED microscopy to increase imaging depth in living brain tissue. bioRxiv, https://doi.org/10.1101/2021.01.05.425408.Search in Google Scholar
Bethge, P., Chereau, R., Avignone, E., Marsicano, G., and Nägerl, U.V. (2013). Two-photon excitation STED microscopy in two colors in acute brain slices. Biophys. J.104, 778–785, https://doi.org/10.1016/j.bpj.2012.12.054.Search in Google Scholar PubMed PubMed Central
Chéreau, R., Saraceno, G.E., Angibaud, J., Cattaert, D., and Nägerl, U.V. (2017). Superresolution imaging reveals activity-dependent plasticity of axon morphology linked to changes in action potential conduction velocity. Proc. Natl. Acad. Sci. U. S. A.114, 1401–1406, https://doi.org/10.1073/pnas.1607541114.Search in Google Scholar PubMed PubMed Central
DeFelipe, J. (2009). Cajal’s place in the history of neuroscience. Encyclopedia of Neuroscience. Squire, L.R., ed. (Oxford: Academic Press), pp. 497–507.10.1016/B978-008045046-9.00989-XSearch in Google Scholar
Fürstenberg, A. and Heilemann, M. (2013). Single-molecule localization microscopy-near-molecular spatial resolution in light microscopy with photoswitchable fluorophores. Phys. Chem. Chem. Phys.15, 14919–14930, https://doi.org/10.1039/c3cp52289j.Search in Google Scholar PubMed
Gu, L., Kleiber, S., Schmid, L., Nebeling, F., Chamoun, M., Steffen, J., Wagner, J., and Fuhrmann, M. (2014). Long-term in vivo imaging of dendritic spines in the hippocampus reveals structural plasticity. J. Neurosci.34, 13948–13953, https://doi.org/10.1523/jneurosci.1464-14.2014.Search in Google Scholar
Holtmaat, A., Wilbrecht, L., Knott, G.W., Welker, E., and Svoboda, K. (2006). Experience-dependent and cell-type-specific spine growth in the neocortex. Nature441, 979–983, https://doi.org/10.1038/nature04783.Search in Google Scholar PubMed
Inavalli, V.V.G.K., Lenz, M.O., Butler, C., Angibaud, J., Compans, B., Levet, F., Tønnesen, J., Rossier, O., Giannone, G., Thoumine, O., et al.. (2019). A super-resolution platform for correlative live single-molecule imaging and STED microscopy. Nat. Methods16, 1263–1268. https://doi.org/10.1038/s41592-019-0611-8.Search in Google Scholar PubMed
Nägerl, U.V., Willig, K.I., Hein, B., Hell, S.W., and Bonhoeffer, T. (2008). Live-cell imaging of dendritic spines by STED microscopy. Proc. Natl. Acad. Sci. U. S. A.105, 18982–18987, https://doi.org/10.1073/pnas.0810028105.Search in Google Scholar PubMed PubMed Central
Pfeiffer, T., Poll, S., Bancelin, S., Angibaud, J., Inavalli, V.K., Keppler, K., Mittag, M., Fuhrmann, M., and Nägerl, U.V. (2018). Chronic 2P-STED imaging reveals high turnover of dendritic spines in the hippocampus in vivo. Elife7, https://doi.org/10.7554/elife.34700.Search in Google Scholar PubMed PubMed Central
Puthukodan, S., Murtezi, E., Jacak, J., and Klar, T.A. (2020). Localization STED (LocSTED) microscopy with 15 nm resolution. Nanophotonics9, 783–792, https://doi.org/10.1515/nanoph-2019-0398.Search in Google Scholar
Rodríguez, C. and Ji, N. (2018). Adaptive optical microscopy for neurobiology. Curr. Opin. Neurobiol.50, 83–91, https://doi.org/10.1016/j.conb.2018.01.011.Search in Google Scholar PubMed PubMed Central
Stolp, B., Thelen, F., Ficht, X., Altenburger, L.M., Ruef, N., Inavalli, V.V.G.K., Germann, P., Page, N., Moalli, F., Raimondi, A., et al.. (2020). Salivary gland macrophages and tissue-resident CD8. Sci. Immunol.5, https://doi.org/10.1126/sciimmunol.aaz4371.Search in Google Scholar PubMed
Tønnesen, J., Inavalli, V.V.G.K., and Nägerl, U.V. (2018). Super-resolution imaging of the extracellular space in living brain tissue. Cell172, 1108–1121, e1115.10.1016/j.cell.2018.02.007Search in Google Scholar PubMed
Tønnesen, J., Katona, G., Rózsa, B., and Nägerl, U.V. (2014). Spine neck plasticity regulates compartmentalization of synapses. Nat. Neurosci.17, 678–685.10.1038/nn.3682Search in Google Scholar PubMed
Tønnesen, J., Nadrigny, F., Willig, K.I., Wedlich-Söldner, R., and Nägerl, U.V. (2011). Two-color STED microscopy of living synapses using a single laser-beam pair. Biophys. J.101, 2545–2552.10.1016/j.bpj.2011.10.011Search in Google Scholar PubMed PubMed Central
Urban, N.T., Willig, K.I., Hell, S.W., and Nägerl, U.V. (2011). STED nanoscopy of actin dynamics in synapses deep inside living brain slices. Biophys. J.101, 1277–1284, https://doi.org/10.1016/j.bpj.2011.07.027.Search in Google Scholar PubMed PubMed Central
Xie, L., Kang, H., Xu, Q., Chen, M.J., Liao, Y., Thiyagarajan, M., O’Donnell, J., Christensen, D.J., Nicholson, C., Iliff, J.J., et al.. (2013). Sleep drives metabolite clearance from the adult brain. Science342, 373–377, https://doi.org/10.1126/science.1241224.Search in Google Scholar PubMed PubMed Central
Yuste, R. (2013). Electrical compartmentalization in dendritic spines. Annu. Rev. Neurosci.36, 429–449, https://doi.org/10.1146/annurev-neuro-062111-150455.Search in Google Scholar PubMed
© 2021 Walter de Gruyter GmbH, Berlin/Boston