title: Photomarking Relocalization Technique for Correlated Two-Photon and Electron Microcopy Imaging of Single Stimulated Synapses.
abstract: Synapses learn and remember by persistent modifications of their internal structures and composition but, due to their small size, it is difficult to observe these changes at the ultrastructural level in real time. Two-photon fluorescence microscopy (2PM) allows time-course live imaging of individual synapses but lacks ultrastructural resolution. Electron microscopy (EM) allows the ultrastructural imaging of subcellular components but cannot detect fluorescence and lacks temporal resolution. Here, we describe a combination of procedures designed to achieve the correlated imaging of the same individual synapse under both 2PM and EM. This technique permits the selective stimulation and live imaging of a single dendritic spine and the subsequent localization of the same spine in EM ultrathin serial sections. Landmarks created through a photomarking method based on the 2-photon-induced precipitation of an electrodense compound are used to unequivocally localize the stimulated synapse. This technique was developed to image, for the first time, the ultrastructure of the postsynaptic density in which long-term potentiation was selectively induced just seconds or minutes before, but it can be applied for the study of any biological process that requires the precise relocalization of micron-wide structures for their correlated imaging with 2PM and EM.
authors.affiliation: RIKEN-MIT Neuroscience Research Center, Massachusetts Institute of Technology, Cambridge, MA, USA. mbosch@mit.edu.|The Picower Institute for Learning and Memory, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA. mbosch@mit.edu.|Institute for Bioengineering of Catalonia, Barcelona, Spain. mbosch@mit.edu.
authors.forename: Miquel
authors.initials: M
authors.lastname: Bosch
authors.affiliation: The Picower Institute for Learning and Memory, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA.
authors.forename: Jorge
authors.initials: J
authors.lastname: Castro
authors.affiliation: The Picower Institute for Learning and Memory, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA.
authors.forename: Mriganka
authors.initials: M
authors.lastname: Sur
authors.affiliation: RIKEN-MIT Neuroscience Research Center, Massachusetts Institute of Technology, Cambridge, MA, USA.|The Picower Institute for Learning and Memory, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA.|Brain Science Institute, RIKEN, Wako, Saitama, Japan.|Saitama University Brain Science Institute, Saitama University, Saitama, Japan.|School of Life Science, South China Normal University, Guangzhou, China.|Department of Pharmacology, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan.
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Hayashi
mesh_terms: D000818:Animals; D049229:Dendritic Spines / Q000378:metabolism / Q000648:ultrastructure; D017930:Genes, Reporter; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D051379:Mice; D008822:Mice, Transgenic; D008854:Microscopy, Electron / Q000379:methods*; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence / Q000379:methods; D017966:Pyramidal Cells / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D013569:Synapses / Q000378:metabolism* / Q000648:ultrastructure*
keywords: Correlated imaging; DAB; Dendritic spine; Photobranding; Photoetching; Photomarking; Postsynaptic density; Serial-section transmission electron microscopy; Synapse; Time-lapse live two-photon fluorescence microscopy