Particle-based phasor-FLIM-FRET resolves protein-protein interactions inside single viral particles

Area of Science:

• Biophysics
• Microscopy
• Molecular Biology

Background:

• Fluorescence Lifetime Imaging Microscopy (FLIM) offers contrast in fluorescence imaging by analyzing nanosecond lifetime patterns.
• Single-molecule FLIM often faces low signal intensities, complicating quantitative analysis of molecular populations and Förster Resonance Energy Transfer (FRET).

Purpose of the Study:

• To investigate the benefits of object localization and the phasor approach in FLIM for FRET analysis at the single-particle level.
• To assess the feasibility of particle-based phasor-FLIM-FRET for studying protein-protein interactions in low-signal environments like viral particles.

Main Methods:

• Simulations were used to determine the photon requirements for accurate phasor signature determination.
• Particle-based phasor-FLIM-FRET was applied to immobilized DNA molecules and HIV-1 viral particles.
• Fluorescent protein probes were quantitatively compared for brightness, lifetime, and photostability in viral particles.

Main Results:

• Approximately 300 photons per particle are sufficient for accurate phasor analysis.
• Particle-based phasor-FLIM-FRET accurately estimated fluorescence lifetimes and FRET efficiencies from single DNA molecules.
• eGFP, mTurquoise2, and mScarlet were identified as optimal FRET probes for HIV-1 integrase fusion.
• Absolute FRET efficiency of HIV-1 integrase oligomers in viral particles was determined.

Conclusions:

• Object localization combined with the phasor approach enhances FLIM-based FRET analysis for single particles, especially those with low signal intensities.
• Particle-based phasor-FLIM-FRET is a valuable tool for investigating molecular interactions and oligomerization states in complex biological systems like viral particles.