Imaging intracellular protein interactions/activity in neurons using 2-photon fluorescence lifetime imaging microscopy.

Through the decades, 2-photon fluorescence microscopy has allowed visualization of microstructures, such as synapses, with high spatial resolution in deep brain tissue. However, signal transduction, such as protein activity and protein-protein interaction in neurons in tissues and in vivo, has remained elusive because of the technical difficulty of observing biochemical reactions at the level of subcellular resolution in light-scattering tissues. Recently, 2-photon fluorescence microscopy combined with fluorescence lifetime imaging microscopy (2pFLIM) has enabled visualization of various protein activities and protein-protein interactions at submicrometer resolution in tissue with a reasonable temporal resolution. Thus far, 2pFLIM has been extensively applied for imaging kinase and small GTPase activation in dendritic spines of hippocampal neurons in slice cultures. However, it has been recently applied to various subcellular structures, such as axon terminals and nuclei, and has increased our understanding of spatially organized molecular dynamics. One of the future directions of 2pFLIM utilization is to combine various optogenetic tools for manipulating protein activity. This combination allows the activation of specific proteins with light and visualization of its readout as the activation of downstream molecules. Here, we have introduced the recent application of 2pFLIM for neurons and present the utilization of a new optogenetic tool in combination with 2pFLIM.