KENBIKYO Vol.50▶No.2 2015

Quantitative Measurements of the Activity of Signaling Molecules by Two-Photon Fluorescence Lifetime Imaging Microscopy

Hideji Murakoshi

Abstract: Recent popularization of 2-photon fluorescence microscopy has enabled us to image cellular structures such as synapses located in deep tissue at the high spatial resolution. However, signal transduction mechanisms in synapse has been still elusive because of the lack of techniques to visualize protein activities or protein-protein interactions. Recently, the improvement of 2-photon fluorescence lifetime imaging microscopy (2pFLIM) to visualize the Förster resonance energy transfer (FRET) has overcame such a difficulty and has enabled us to visualize biochemical reactions. Using this technique, we have recently succeeded in imaging the activity of small GTPases. Here I introduce the principle of the 2pFLIM for monitoring intracellular protein activities and protein-protein interactions with an example: detecting small GTPase (Cdc42 and RhoA) activity in dendrites and synapses of hippocampal neurons in brain slices.

Key words: 2-photon microscope, fluorescence lifetime imaging, small GTPase protein