Supercritical angle fluorescence (SAF) is a near-field collection method that has surface sensitivities similar to or better than near-field excitation techniques like TIRF and waveguide based excitation. SAF is emitted by fluorophores that are a few hundred nanometers away from an interface, above the critical angle, into the higher index material. SAF decreases exponentially with increasing distance from the interface and is therefore more sensitive to molecules near the surface. Although a lot of research has used SAF for biosensing and microscopy, the angular dependence of SAF on both the surface and bulk fluorescence contributions hasn’t been experimentally studied. We present a method that measures the surface selectivity of SAF in the presence of bulk fluorophores. Two different fluorophores were used. One was bound to the surface and the other was suspended in the bulk. The spectrum was measured at discrete points in the back focal plane (BFP) and the contribution of the two fluorophores was extracted from it. The results of the experiment show the highest signal-to-noise ratio in the region just above the critical angle of 61.31º because of the higher signal intensity. However, for experiments where bulk exclusion is important, we observe the highest signal-to-bulk ratio at angles above 68˚ for a glass-water interface. Understanding the angular dependence on the sensitivity of a SAF biosensor enables tuning the collection angles towards specific applications and could lead to the creation of smaller, more sensitive devices.
Finub James Shirley, Pieter Neutens, Rita Vos, Md. Mahmud-Ul-Hasan, Niels Verellen, and Pol Van Dorpe, "Angular dependence of bulk fluorescence noise in supercritical angle fluorescence (Conference Presentation)," Proc. SPIE 10506, Nanoscale Imaging, Sensing, and Actuation for Biomedical Applications XV, 105060P (Presented at SPIE BiOS: January 31, 2018; Published: 15 March 2018); https://doi.org/10.1117/12.2289951.5752194732001.
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