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Synapse Development pp 185–214Cite as

Photomarking Relocalization Technique for Correlated Two-Photon and Electron Microcopy Imaging of Single Stimulated Synapses

Part of the Methods in Molecular Biology book series (MIMB,volume 1538)

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.

Key words

  • Correlated imaging
  • Time-lapse live two-photon fluorescence microscopy
  • Serial-section transmission electron microscopy
  • Dendritic spine
  • Synapse
  • Postsynaptic density
  • Photomarking
  • Photoetching
  • Photobranding
  • DAB

These two authors contributed equally to this chapter.

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Acknowledgments

This work was supported by a “Beatriu de Pinós” fellowship (AGAUR, “Generalitat de Catalunya”), the FRAXA Foundation, a Marie Curie Reintegration Grant (H2020-MSCA-IF) (to M.B.), the “Fundación Caja Madrid” (to J.C.), the Anne Punzak Marcus Fund (to M.S.), RIKEN, a NIH grant (R01DA17310), a Grant-in-Aid for Scientific Research (A), and a Grant-in-Aid for Scientific Research on Innovative Area “Foundation of Synapse and Neurocircuit Pathology” from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Human Frontier Science Program, and The Key Recruitment Program of High-end Foreign Experts of the Administration of Foreign Experts Affairs of Guangdong Province (to Y.H.). Conflict of interest statement: Y.H. is partly supported by Takeda Pharmaceutical Co. Ltd. and Fujitsu Laboratories.

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    Authors and Affiliations

    1. RIKEN-MIT Neuroscience Research Center, Massachusetts Institute of Technology, Cambridge, MA, USA

      Miquel Bosch & Yasunori Hayashi

    2. The Picower Institute for Learning and Memory, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA

      Miquel Bosch, Jorge Castro, Mriganka Sur & Yasunori Hayashi

    3. Institute for Bioengineering of Catalonia, Barcelona, Spain

      Miquel Bosch

    4. Brain Science Institute, RIKEN, Wako, Saitama, Japan

      Yasunori Hayashi

    5. Saitama University Brain Science Institute, Saitama University, Saitama, Japan

      Yasunori Hayashi

    6. School of Life Science, South China Normal University, Guangzhou, China

      Yasunori Hayashi

    7. Department of Pharmacology, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan

      Yasunori Hayashi

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    1. Miquel Bosch
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    2. Jorge Castro
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    4. Yasunori Hayashi
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    Correspondence to Miquel Bosch .

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    1. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA

      Alexandros Poulopoulos

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    Bosch, M., Castro, J., Sur, M., Hayashi, Y. (2017). Photomarking Relocalization Technique for Correlated Two-Photon and Electron Microcopy Imaging of Single Stimulated Synapses. In: Poulopoulos, A. (eds) Synapse Development. Methods in Molecular Biology, vol 1538. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6688-2_14

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    • DOI: https://doi.org/10.1007/978-1-4939-6688-2_14

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