Localizing protein-protein interactions in living cells using fluorescence lifetime imaging microscopy

Area of Science:

• Life Sciences
• Biotechnology
• Microscopy

Background:

• Fluorescence lifetime imaging has seen significant advancements in the last decade.
• Applications span fundamental biological research to clinical diagnostics.
• Fluorescence lifetime imaging microscopy (FLIM) is crucial for observing dynamic cellular signaling, including protein-protein interactions.

Purpose of the Study:

• To describe the calibration of time-correlated single-photon counting (TCSPC) and frequency domain (FD) FLIM systems.
• To detail the acquisition and analysis of FLIM-FRET data.
• To investigate protein-protein interactions within living cells using FLIM-FRET.

Main Methods:

• Calibration of TCSPC-FLIM and FD-FLIM systems.
• Acquisition of FLIM-FRET data from living cells.
• Analysis of FLIM-FRET data to assess protein-protein interactions.

Main Results:

• Established protocols for FLIM system calibration.
• Demonstrated methods for FLIM-FRET data acquisition and analysis.
• Provided a framework for studying protein-protein interactions in vivo.

Conclusions:

• FLIM is a powerful technique for studying dynamic biological processes.
• Accurate calibration and data analysis are essential for reliable FLIM-FRET results.
• This work facilitates the investigation of protein-protein interactions in living systems.