FLIM-FRET as a Molecular Filter for Membrane-Induced Aggregation

Area of Science:

• Neuroscience
• Biophysics
• Cell Biology

Background:

• Membrane binding of alpha-synuclein (aSyn) is implicated in early neuronal aggregation.
• Accurate live-cell measurement of membrane-proximal aSyn aggregation remains a significant challenge.

Purpose of the Study:

• To develop a high-specificity method for quantifying membrane-proximal alpha-synuclein aggregation in live neurons.
• To establish per-cell metrics for membrane proximity and aggregation states.

Main Methods:

• Combined three-channel fluorescence lifetime imaging microscopy (FLIM) with Förster resonance energy transfer (FRET).
• Utilized a hierarchical expectation-maximization (EM) algorithm to estimate per-cell lifetimes and population fractions.
• Validated the method using Monte Carlo simulations and experimental neuronal data.

Main Results:

• Successfully resolved changes in membrane-proximal aggregation and aggregate-associated lifetimes in neurons.
• Demonstrated improved accuracy in estimating cellular aggregation states under realistic conditions.
• Revealed previously unattainable insights into membrane-proximal aggregation effects.

Conclusions:

• The developed FLIM-FRET approach with hierarchical analysis provides robust cell-level metrics for aggregation studies.
• This method overcomes limitations of pixel-by-pixel or whole-cell averaging, enabling reliable comparisons across experimental conditions.
• The framework is broadly applicable for studying protein aggregation and membrane interactions beyond alpha-synuclein.