Optimal Detection of Fusion Pore Dynamics Using Polarized Total Internal Reflection Fluorescence Microscopy

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Membrane fusion pore dynamics are crucial for cellular processes like hormone release.
• Understanding fusion pore mechanisms is limited by the lack of high-resolution, single-pore assays.
• Existing Total Internal Reflection Fluorescence (TIRF) microscopy has signal complexities hindering analysis.

Purpose of the Study:

• To develop a novel polarization-controlled TIRF (pTIRF) microscope for high-resolution membrane fusion studies.
• To investigate the dynamics of fusion pores during vesicle fusion events.
• To analyze lipid transfer kinetics and fusion pore openness.

Main Methods:

• Constructed a custom pTIRF microscope with controllable excitation polarization.
• Monitored fusion of proteoliposomes (vSUVs) with lipid bilayers (tSBL) using single-molecule sensitivity.
• Employed an improved algorithm to estimate vesicle size and fusion pore openness.

Main Results:

• pTIRF microscopy enabled sensitive detection and analysis of single membrane fusion events.
• Identified optimal excitation polarization for enhanced fluorescence signal during fusion.
• Observed that fusion pore flickering, not diffusion, likely limits lipid transfer, with weak correlation to vesicle size.

Conclusions:

• The developed pTIRF microscope overcomes limitations of standard TIRF for studying membrane fusion.
• Fusion pore flickering plays a significant role in regulating cargo release during vesicle fusion.
• This approach provides a versatile tool for investigating diverse membrane fusion processes.