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

Supercritical angle fluorescence (SAF) detection combines the axial discrimination and exquisite signal-to-noise ratio of total internal reflection fluorescence (TIRF) with the lateral discrimination and convenience of confocal excitation. This combination makes SAF ideal for fluorescence correlation spectroscopy (FCS) on membranes and other structures in close proximity to the coverslip. Here we report a straightforward modification of a commercial microscope to implement SAF FCS and demonstrate in both model supported lipid bilayers and cellular systems that this approach shows an increase in signal from membrane-bound fluorophores relative to fluorophores in solution, benchmarked against line-scanning FCS. SAF FCS allowed us to demonstrate that activation of the T cell receptor resulted in the recruitment of the kinase Lck to the plasma membrane as well as a reduction in Lck mobility within the membrane.