Optimization of resonant dielectric multilayer for enhanced fluorescence imaging

In the aim of improving the sensitivity in objective-based total internal reflection fluorescence microscopy (ob-TIRF-M), we recently investigated the use of resonant dielectric multilayer (DM) coated coverslips while considering the experimental constraints of commercial ob-TIRF-M. The imaginary part of the refractive index k of the DM top layer material was then shown to be a key parameter to control during the DM optimization in order to limit the discrepancies between theory and experiment. We therefore report here on the development, fabrication and implementation of a specific top layer with a controlled k. This latter was first investigated as a single layer to accurately define its optical properties while adjusting the stoichiometric conditions during deposition. The DM optimization method was then fully revised to include its optical parameters. By doing so, the optimization was freed from the strong dependence on k and we observed a good agreement between the expected and measured fluorescence signal enhancement. This was illustrated by numerically calculating the fluorescence signal enhancement as a function of the top layer k and by comparing it to fluorescent beads imaging over three DM of different k.