Virtual Supercritical Angle Fluorescence Microscopy for Real Time Membrane Imaging

We present an alternative full field imaging technique based on Supercritical Angle Fluorescence (SAF). When fluorescent emitters are placed in the vicinity of the glass slide, their near-field components become propagative at supercritical angles. This supercritical emission decays sharply the fluorophore/surface distance d with a characteristic decay length of about 100 nm. Selecting the supercritical emission thus provides an efficient way to realize spatial filtering. This technique has numerous advantages over techniques based on excitation confinement like TIRFM. In particular, it avoids the major drawbacks of the loss of confinement due to light scattering. Standard light sources, with a homogeneous lighting, can be used instead of the usually required lasers.