Measuring membrane association and protein diffusion within membranes with supercritical angle fluorescence...

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Total internal reflection fluorescence (TIRF) offers high signal-to-noise but lacks lateral discrimination.
• Confocal microscopy provides lateral discrimination but has limited signal efficiency for membrane studies.
• Supercritical angle fluorescence (SAF) detection merges TIRF's axial discrimination with confocal's lateral discrimination.

Purpose of the Study:

• To implement and validate SAF detection for fluorescence correlation spectroscopy (FCS) on a commercial microscope.
• To assess the signal enhancement of SAF FCS for membrane-bound fluorophores compared to solution-based fluorophores.
• To investigate T cell receptor (TCR) activation dynamics and Lck kinase recruitment and mobility in cellular membranes.

Main Methods:

• Modification of a commercial microscope to incorporate SAF detection.
• Application of SAF FCS to model supported lipid bilayers and live cellular systems.
• Benchmarking SAF FCS performance against line-scanning FCS.
• Monitoring T cell receptor activation and Lck kinase behavior.

Main Results:

• SAF FCS demonstrated increased signal from membrane-bound fluorophores compared to solution fluorophores.
• The method was validated in both model lipid bilayers and cellular environments.
• SAF FCS successfully observed recruitment of Lck kinase to the plasma membrane upon TCR activation.
• A reduction in Lck mobility within the membrane was observed following TCR activation.

Conclusions:

• SAF FCS is a straightforward and effective technique for studying membrane dynamics.
• This method provides enhanced signal for membrane-bound molecules, improving resolution in FCS.
• SAF FCS revealed key insights into TCR-mediated signaling, including Lck recruitment and altered mobility.