Optogenetic Imaging of Protein Activity Using Two-Photon Fluorescence Lifetime Imaging Microscopy

Area of Science:

• Cellular Biology
• Biophysics
• Neuroscience

Background:

• Spatiotemporal dynamics of cellular proteins, including interactions and conformational changes, are crucial for cellular functions like synaptic plasticity and cell division.
• Visualizing protein activity in living cells and tissues at high spatiotemporal resolution is essential for understanding signal transduction mechanisms.

Purpose of the Study:

• To introduce the principles of two-photon fluorescence lifetime imaging microscopy with Förster Resonance Energy Transfer (2pFLIM-FRET).
• To highlight the use of chromoproteins for imaging intracellular protein activities and interactions.
• To discuss applications of 2pFLIM-FRET in biological research.

Main Methods:

• Utilizes optogenetic probes, specifically fluorescent proteins, in conjunction with Förster Resonance Energy Transfer (FRET) techniques.
• Employs two-photon fluorescence lifetime imaging microscopy (2pFLIM) for FRET detection.
• Focuses on imaging within subcellular compartments of living cells, particularly deep within tissues like brain slices.

Main Results:

• 2pFLIM-FRET enables monitoring of FRET in challenging environments, such as deep brain tissue.
• Chromoproteins can be effectively used with 2pFLIM-FRET for visualizing protein dynamics.
• Demonstrates successful application in imaging actin polymerization in hippocampal neuron synapses and Cdc42 activity in astrocytes.

Conclusions:

• 2pFLIM-FRET is a powerful technique for high-resolution imaging of protein dynamics in living cells within tissues.
• This method provides insights into cellular signaling pathways and protein interactions.
• The presented applications showcase the versatility of 2pFLIM-FRET in neuroscience and cell biology.