Imaging Neuronal Signal Transduction Using Multiphoton FRET-FLIM

  • Paul R. Evans
  • Long Yan
  • Ryohei YasudaEmail author
Part of the Neuromethods book series (NM, volume 148)


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.


Fluorescence lifetime imaging microscopy (FLIM) Two-photon microscopy Fluorescence resonance energy transfer (FRET) Sensor biology Dendritic spine Neuronal signal transduction Spine structural plasticity Long-term potentiation (LTP) 



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.


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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Max Planck Florida Institute for NeuroscienceJupiterUSA

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