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Multiphoton Microscopy pp 111–130Cite as

Imaging Neuronal Signal Transduction Using Multiphoton FRET-FLIM

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Part of the book series: Neuromethods ((NM,volume 148))

Abstract

Synaptic plasticity, the ability of neurons to modulate the strength of specific inputs, is critical for neural circuits to adapt to experience throughout life. In excitatory pyramidal neurons, plasticity is induced by coincident neuronal activity and glutamate release at tiny postsynaptic protrusions called dendritic spines, which initiate the coordinated activity of hundreds of different proteins located in spines and throughout the neuron at distinct temporal phases. Thus, elucidating the spatiotemporal dynamics of individual signaling proteins is critical to refine our understanding of this process. The complex, polarized morphology of neurons can restrict protein activity to small cellular subcompartments, while other signals can spread over long distances, which poses unique challenges to monitoring protein dynamics. Fluorescence resonance energy transfer (FRET) is a useful photophysical phenomenon to visualize signaling in space and time within live cells by measuring the efficiency of energy transfer between two fluorescent proteins. Using two-photon fluorescence lifetime imaging microscopy (2pFLIM) to assay FRET-based signaling sensors permits chronic, high-resolution measurements of discrete neuronal signaling events, even in dense, light-scattering brain slices. Here, we describe the imaging setup required to perform 2pFLIM and highlight its application to decipher the orchestrated signaling underlying the structural plasticity of dendritic spines.

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Acknowledgments

We thank Dr. Lesley Colgan, Dr. Tal Laviv, and other members of the Yasuda lab for thoughtful discussion. We also thank Dr. Nathan Hedrick for contributing to the figures.

Conflict of Interest

Dr. Ryohei Yasuda is the founder of Florida Lifetime Imaging LLC., a company that helps people set up FLIM.

Author information

Authors and Affiliations

  1. Max Planck Florida Institute for Neuroscience, Jupiter, FL, USA

    Paul R. Evans, Long Yan & Ryohei Yasuda

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  1. Paul R. Evans
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  2. Long Yan
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  3. Ryohei Yasuda
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Corresponding author

Correspondence to Ryohei Yasuda .

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Editors and Affiliations

  1. Department of Biomedicine, University of Bergen, Bergen, Norway

    Espen Hartveit

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Evans, P.R., Yan, L., Yasuda, R. (2019). Imaging Neuronal Signal Transduction Using Multiphoton FRET-FLIM. In: Hartveit, E. (eds) Multiphoton Microscopy. Neuromethods, vol 148. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9702-2_6

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  • DOI: https://doi.org/10.1007/978-1-4939-9702-2_6

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9701-5

  • Online ISBN: 978-1-4939-9702-2

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