Two-Photon FRET/FLIM Imaging of Cerebral Neurons

Thomas T Luyben; Jayant Rai; Bingyue Zhou; Hang Li; Kenichi Okamoto

doi:10.1007/978-1-0716-3810-1_4

Two-Photon FRET/FLIM Imaging of Cerebral Neurons

Methods Mol Biol. 2024:2794:33-43. doi: 10.1007/978-1-0716-3810-1_4.

Authors

Thomas T Luyben^{1

2}, Jayant Rai^{1

2}, Bingyue Zhou^{1

2}, Hang Li¹, Kenichi Okamoto^{3

4}

Affiliations

¹ Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada.
² Department of Molecular Genetics, Faculty of Medicine, University of Toronto, Toronto, ON, Canada.
³ Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada. okamoto@lunenfeld.ca.
⁴ Department of Molecular Genetics, Faculty of Medicine, University of Toronto, Toronto, ON, Canada. okamoto@lunenfeld.ca.

PMID: 38630218
DOI: 10.1007/978-1-0716-3810-1_4

Abstract

Two-photon FRET (Förster resonance energy transfer) and FLIM (fluorescence lifetime imaging microscopy) enable the detection of FRET changes of fluorescence reporters in deep brain tissues, which provide a valuable approach for monitoring target molecular dynamics and functions. Here, we describe two-photon FRET and FLIM imaging techniques that allow us to visualize endogenous and optogenetically induced cAMP dynamics in living neurons with genetically engineered FRET-based cAMP reporters.

Keywords: Dendritic spine; FLIM; FRET; Fluorescence reporters; Optogenetics; Photoactivatable adenylyl cyclase (PAC); Synaptic plasticity; Two-photon microscopy; cAMP.

MeSH terms

Fluorescence Resonance Energy Transfer*
Genetic Engineering*
Microscopy, Fluorescence
Neurons
Photons