PIE-FLIM Measurements of Two Different FRET-Based Biosensor Activities in the Same Living Cells

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Genetically encoded biosensors often use Förster resonance energy transfer (FRET) to report on cellular events.
• Quantifying FRET via fluorescence lifetime imaging microscopy (FLIM) provides robust measurements of biosensor conformation changes.
• Studying complex cellular signaling requires simultaneous monitoring of multiple pathways.

Purpose of the Study:

• To demonstrate the simultaneous measurement of two distinct biosensor activities in single living cells using PIE-FLIM.
• To showcase the capability of PIE-FLIM in resolving and quantifying signals from multiple FRET-based biosensors concurrently.
• To highlight the broad applicability of PIE-FLIM for studying complex signaling networks.

Main Methods:

• Utilized pulsed interleaved excitation (PIE) combined with fluorescence lifetime imaging microscopy (FLIM).
• Employed selectable laser wavelengths and synchronized detection gating optimized for specific FRET pairs.
• Applied the technique to simultaneously track cytosolic calcium and protein kinase A activity in living cells.

Main Results:

• Successfully demonstrated the simultaneous measurement of two different biosensor activities within single living cells.
• Showcased the ability of PIE-FLIM to separate and quantify signals from distinct FRET-based biosensors.
• Provided proof-of-principle for simultaneous monitoring of cytosolic calcium and protein kinase A activity.

Conclusions:

• PIE-FLIM is a powerful technique for simultaneously measuring multiple biosensor activities in living cells.
• This method allows for the concurrent study of different cell signaling pathways within the same cell.
• The PIE-FLIM approach offers broad applicability for investigating complex biological signaling events.