Supercritical Angle Fluorescence Microscopy and Spectroscopy

Abstract

Fluorescence detection, either involving propagating or near-field emission, is widely being used in spectroscopy, sensing, and microscopy. Total internal reflection fluorescence (TIRF) confines fluorescence excitation by an evanescent (near) field, and it is a popular contrast generator for surface-selective fluorescence assays. Its emission equivalent, supercritical angle fluorescence (SAF), is comparably less established, although it achieves a similar optical sectioning as TIRF does. SAF emerges when a fluorescing molecule is located very close to an interface and its near-field emission couples to the higher refractive index medium (n₂ >n₁) and becomes propagative. Then, most fluorescence is detectable on the side of the higher-index substrate, and a large fraction of this fluorescence is emitted into angles forbidden by Snell's law. SAF, as well as the undercritical angle fluorescence (UAF; far-field emission) components, can be collected with microscope objectives having a high-enough detection aperture (numerical aperture >n₂) and be separated in the back focal plane by Fourier filtering. The back focal plane image encodes information about the fluorophore radiation pattern, and it can be analyzed to yield precise information about the refractive index in which the emitters are embedded, their nanometric distance from the interface, and their orientation. A SAF microscope can retrieve this near-field information through wide-field optics in a spatially resolved manner, and this functionality can be added to an existing inverted microscope. Here, we describe the potential underpinning of SAF microscopy and spectroscopy, particularly in comparison with TIRF. We review the challenges and opportunities that SAF presents from a biophysical perspective, and we discuss areas in which we see potential.