Super-Resolution Dynamic Imaging of Dendritic Spines Using a Low-Affinity Photoconvertible Actin Probe Ignacio Izeddin 1. , Christian G. Specht 2. , Mickae ¨ l Lelek 3 , Xavier Darzacq 4 , Antoine Triller 2 *, Christophe Zimmer 3 , Maxime Dahan 1 * 1 Laboratoire Kastler Brossel, CNRS UMR 8552, Departments of Physics and Biology, E ´ cole Normale Supe ´rieure, Universite ´ Pierre et Marie Curie-Paris 6, Paris, France, 2 Biologie Cellulaire de la Synapse, E ´ cole Normale Supe ´rieure, Inserm U1024, Paris, France, 3 Institut Pasteur, Groupe Imagerie et Mode ´lisation, CNRS, URA 2582, Paris, France, 4 Re ´ gulation de l’Expression Ge ´ne ´tique, E ´ cole Normale Supe ´rieure, CNRS UMR8541, Paris, France Abstract The actin cytoskeleton of dendritic spines plays a key role in morphological aspects of synaptic plasticity. The detailed analysis of the spine structure and dynamics in live neurons, however, has been hampered by the diffraction-limited resolution of conventional fluorescence microscopy. The advent of nanoscopic imaging techniques thus holds great promise for the study of these processes. We implemented a strategy for the visualization of morphological changes of dendritic spines over tens of minutes at a lateral resolution of 25 to 65 nm. We have generated a low-affinity photoconvertible probe, capable of reversibly binding to actin and thus allowing long-term photoactivated localization microscopy of the spine cytoskeleton. Using this approach, we resolve structural parameters of spines and record their long- term dynamics at a temporal resolution below one minute. Furthermore, we have determined changes in the spine morphology in response to pharmacologically induced synaptic activity and quantified the actin redistribution underlying these changes. By combining PALM imaging with quantum dot tracking, we could also simultaneously visualize the cytoskeleton and the spine membrane, allowing us to record complementary information on the morphological changes of the spines at super-resolution. Citation: Izeddin I, Specht CG, Lelek M, Darzacq X, Triller A, et al. (2011) Super-Resolution Dynamic Imaging of Dendritic Spines Using a Low-Affinity Photoconvertible Actin Probe. PLoS ONE 6(1): e15611. doi:10.1371/journal.pone.0015611 Editor: Vadim E. Degtyar, University of California, United States of America Received September 10, 2010; Accepted November 17, 2010; Published January 17, 2011 Copyright: ß 2011 Izeddin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work is supported by the Fondation pour la Recherche Me ´dicale (FRM), the Fondation Pierre-Gilles de Gennes, and C’Nano Ile de France. I.I. acknowledges the Netherlands Organisation for Scientific Research (NWO) for financial support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: maxime.dahan@lkb.ens.fr (MD); triller@biologie.ens.fr (AT) . These authors contributed equally to this work. Introduction Fluorescence microscopy using genetically encoded fluorescent proteins has greatly advanced our understanding of many functional biological systems over the last decade. However, the precision at which cellular structures can be visualized has been limited by the spatial resolution imposed by the diffraction limit of light (,250 nm). Novel super-resolution imaging methods using photoactivatable proteins or photoswitchable fluorophores bypass this limitation and have the potential to revolutionize the experimental scope of light microscopy [1]. The application of these methods in living cells is far from trivial, although recent work has achieved a remarkable progress in this direction [2–7]. A general problem is the time needed to acquire a super-resolution image and the associated bleaching of fluorophores, which limits the temporal resolution as well as the use of these techniques for long-term imaging. Dendritic spines are small cellular structures that compartmen- talize the sites of excitatory neurotransmission in neurons [8]. The small dimensions of spines (,500 nm in diameter) make super- resolution methods ideally suited to image the morphology of spines at much greater detail than that achieved by conventional fluorescence microscopy. The structure of dendritic spines is defined by the F-actin cytoskeleton and can undergo fast dynamic morphological changes that are believed to contribute to the plasticity of synaptic transmission [9]. While physiological and morphological aspects of synaptic plasticity are under certain conditions independent from one another [10], the enhancement of synaptic transmission by long-term potentiation (LTP) appears to be generally associated with an increase in spine volume [4,11]. In line with these observations, the polymerization state and the dynamic properties of the actin cytoskeleton in dendritic spines have been shown to change during synaptic plasticity [12,13]. These studies aimed at identifying different actin pools by distinguishing between populations of actin molecules using photoactivatable fluorophores and FRET, respectively. Morpho- logical changes associated with synaptic activity occur on relatively slow time scale of tens of minutes [4], however the tools to image these changes continuously at high spatial resolution are limited. Therefore the goal of our study was to develop a strategy that would enable us to visualize the dynamic changes of the spine morphology for long periods. Recent studies have combined the use of photoactivatable probes and single particle tracking (SPT) to study the kinetics of the actin cytoskeleton of spines [14,15]. The short-range motion of individual actin molecules in these experiments suggested a complex meshwork of actin filaments in dendritic spines, in line with observations made by electron microscopy (EM) [16,17]. PLoS ONE | www.plosone.org 1 January 2011 | Volume 6 | Issue 1 | e15611