Label-free microrefractometry of interfacial processes using fluorescent smart coverslips

Molecular dipoles near interfaces emit highly directional radiation due to near-field interactions, making surface-bound fluorophores sensitive probes of local physicochemical changes. We introduce "smart coverslips," standard microscope substrates, which are stably coated with uniform, brightly fluorescent nanobead films, that exploit refractive-index-dependent shifts in the radiation pattern for sensitive microrefractometry in small volumes. Supercritical angle fluorescence refractometry uses single back focal plane images to allow us real-time refractive index sensing and nanometric thin-film height measurements without the need for multi-angle or multi-wavelength acquisition. Our fast, label-free, and noninvasive 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.