Supercritical Angle Fluorescence Characterization Using Spatially Resolved Fourier Plane Spectroscopy.

Most fluorescent immunoassays require a wash step prior to read-out due to the otherwise overwhelming signal of the large number of unbound (bulk) fluorescent molecules that dominate over the signal from the molecules of interest, usually bound to a substrate. Supercritical angle fluorescence (SAF) sensing is one of the most promising alternatives to total internal reflection fluorescence for fluorescence imaging and sensing. However, detailed experimental investigation of the influence of collection angle on the SAF surface sensitivity, i.e., signal to background ratio (SBR), is still lacking. In this Letter, we present a novel technique that allows to discriminate the emission patterns of free and bound fluorophores simultaneously by collecting both angular and spectral information. The spectrum was probed at multiple positions in the back focal plane using a multimode fiber connected to a spectrometer and the difference in intensity between two fluorophores was used to calculate the SBR. Our study clearly reveals that increasing the angle of SAF collection enhances the surface sensitivity, albeit at the cost of decreased signal intensity. Furthermore, our findings are fully supported by full-field 3D simulations.