Application of phasor plot and autofluorescence correction for study of heterogeneous cell population

Area of Science:

• Cellular biology
• Biophysics
• Microscopy

Background:

• Protein-protein interactions are crucial cellular processes.
• Fluorescence resonance energy transfer (FRET) coupled with fluorescence lifetime imaging microscopy (FLIM) is a powerful tool for studying these interactions.
• Quantitative FRET analysis can be challenging due to cellular heterogeneity and complex photophysical phenomena.

Purpose of the Study:

• To develop and validate quantitative methods for FRET-FLIM analysis in complex cellular environments.
• To accurately measure FRET efficiency and binding affinity in heterogeneous cell populations.
• To investigate the interaction between Toll-like receptor 4 (TLR4) and a decoy peptide.

Main Methods:

• Application of multifrequency phasor plots for visualizing FRET-FLIM data.
• Development of a lifetime background correction method.
• Time-resolved analysis using a biexponential model.
• Utilizing Toll-like receptor 4 fused to Cerulean (TLR4-Cer) as a donor and Bodipy TMRX-labeled 4BB peptide (4BB-BTX) as an acceptor.

Main Results:

• Successful visualization of FRET-FLIM data using phasor displays.
• Demonstration of effective lifetime background correction.
• Determination of absolute FRET efficiency.
• Estimation of binding affinity between TLR4-Cer and 4BB-BTX in heterogeneous HeLa cells.

Conclusions:

• The developed FRET-FLIM analytical approaches enable robust quantitative analysis even in challenging biological samples.
• The study provides insights into the binding characteristics of TLR4 and its decoy peptide.
• This methodology can be broadly applied to study other protein-protein interactions in complex cellular systems.