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Investigating Molecular Diffusion Inside Small Neuronal Compartments with Two-Photon Fluorescence Correlation Spectroscopy

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Single Molecule Microscopy in Neurobiology

Part of the book series: Neuromethods ((NM,volume 154))

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Abstract

Molecular mobility within cells is regulated by cytoplasmic structures and cell geometries. Conversely, intricate cellular functions may depend on heterogeneity and compartment-specific regulation of molecular dynamics. Precise measurements of molecular mobility within neuronal cells are challenging due to their highly complex morphology and heterogeneity in the intracellular environment. Fluorescence correlation spectroscopy (FCS) is a sensitive method for measuring diffusion coefficients of fluorescent molecules. Since FCS relies on molecular motion across a detection volume defined by the optical resolution (<1 fL), FCS is an appropriate method for measurements inside small cellular compartments. Here, we describe basic equipment for two-photon FCS and procedures of measurement and data analysis in cells expressing fluorescent proteins. We also show the data from two-photon FCS measurements inside dendritic shafts or spines of cultured hippocampal neurons and discuss the effect of cellular geometry on measurements of small neuronal compartments.

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Acknowledgments

This study was supported by Grants-in-Aid for Scientific Research (17H01387 and 18H04727), Core Research for Evolutional Science and Technology from the Japan Science and Technology Agency (JPMJCR14W2), the Project for Elucidating and Controlling Mechanisms of Aging and Longevity from the Japan Agency for Medical Research and Development (17gm5010003), the Tokyo Center for Integrative Science of Human Behavior (CiSHuB) to S.O., and a Grant-in-Aid for JSPS Fellows (13J03845) to K.O.

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Authors and Affiliations

  1. Department of Cellular Neurobiology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

    Kazuki Obashi & Shigeo Okabe

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  1. Kazuki Obashi
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  2. Shigeo Okabe
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Correspondence to Shigeo Okabe .

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Editors and Affiliations

  1. Laboratory of Cellular and Molecular Neurobiology, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan

    Nobuhiko Yamamoto

  2. Laboratory for Cell Polarity Regulation, RIKEN Center for Biosystems Dynamics Research, Suita, Osaka, Japan

    Yasushi Okada

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Obashi, K., Okabe, S. (2020). Investigating Molecular Diffusion Inside Small Neuronal Compartments with Two-Photon Fluorescence Correlation Spectroscopy. In: Yamamoto, N., Okada, Y. (eds) Single Molecule Microscopy in Neurobiology . Neuromethods, vol 154. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0532-5_15

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  • DOI: https://doi.org/10.1007/978-1-0716-0532-5_15

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0531-8

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