Label-Free Microrefractometry of Interfacial Processes Using Fluorescent Smart Coverslips

Molecular dipoles near interfaces emit highly directional radiation due to near-field inter-actions, making surface-bound fluorophores sensitive probes of local physicochemical changes. We intro-duce "smart coverslips", standard microscope substrates, which are stably coated with uniform, brightly fluorescent nano-bead films, that exploit refractive-index-dependent shifts in the radiation pattern for sensitive micro-refractometry in small volumes. Supercritical-angle fluorescence refractometry uses single back-focal-plane images to allow us real-time RI sensing and nanometric thin-film height measurements without the need for multi-angle or multi-wavelength acquisition. Our fast, label-free, and non-invasive approach allows measurements of thin-film properties and monitoring of interfacial dynamics on a standard inverted microscope and is broadly applicable to nano-biophotonics, chemical sensing, and in-situ materials analysis. Applied to membrane biophysics, these findings establish a versatile approach for resolving the nanoscale organization of membrane-proximal or -associated structures.