A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Genetically encoded fluorescent protein (FP)-based biosensors are vital for monitoring cellular events in living systems.
• Existing FP biosensors, designed for one-photon excitation, present challenges for two-photon laser-scanning microscopy (TPLSM) due to broad excitation and unclear photobleaching.
• Implementing Förster resonance energy transfer (FRET)-based biosensors in TPLSM requires optimized protocols.

Purpose of the Study:

• To develop and present a simplified protocol for utilizing FRET-based biosensors with TPLSM.
• To enable efficient and reliable FRET measurements in live cells and tissues using TPLSM.
• To overcome the limitations of current FP biosensors in TPLSM applications.

Main Methods:

• Optimization of TPLSM systems using FRET standards expressed in living cells.
• Determination of spectral bleed-through (SBT) and validation of FRET measurements.
• Experimental application of the protocol using a modified A kinase activity reporter (AKAR) biosensor in cultured cells and mouse hepatocytes.

Main Results:

• A streamlined protocol for FRET biosensor application in TPLSM was established.
• The method allows for accurate FRET measurements and system optimization.
• Successful imaging of biosensor activity was achieved in both cell cultures and in vivo liver hepatocytes.

Conclusions:

• This protocol simplifies the use of FRET biosensors with TPLSM, making advanced live-cell imaging more accessible.
• The developed method facilitates the study of cellular dynamics in complex biological systems.
• The protocol can be implemented within a day in standard TPLSM laboratories.