We present the optimization of dielectric multilayers (DM) for improved total internal reflection fluorescence sensitivity. The desired resonances require a design that achieves a field enhancement and an angular tolerance of the resonance in the order of the illumination divergence. We studied the effect of the imaginary part of the refractive index k of the DM top layer on the fluorescence enhancement. We established a protocol to fabricate a single layer with a controlled k and fabricated three structures of various k. We tested the concept on fluorescent beads and observed a good agreement with the predicted fluorescence enhancement.
TIRF-Microscopy is widely used in cell biology to monitor dynamic biomolecular events occurring at the cell membranes. However, this technic is limited by weak fluorescence signal and background noise degrading the spatial resolution. We present high sensitive fluorescent TIRF-M using glass coverslips coated with one-dimensional photonic crystal (1DPC). The latter is made of dielectric multilayers optimized to generate a large field enhancement under TIR illumination at its free interface. Using a TIRF-microscope, we successfully demonstrate the capability of these substrates to enhance the fluorescence signal as compared to classical glass coverslips over fluorescently labeled HIV-1 and SARS-CoV-2 virus-like-particles imaging.
Total Internal Reflection Fluorescence (TIRF) Microscopy is widely used in cell biology to monitor dynamic biomolecular events occurring at the plasma membrane in living cells such as virus assemblies and particle budding. However, these studies are limited by the weak fluorescence signal and background noise degrading the spatial resolution. We present highly sensitive fluorescent TIRF imaging using classical glass coverslips coated with a resonant multi-dielectric thin film. Such dielectric multilayer is optimized to generate a large field enhancement under TIR illumination at the free interface. However, dielectric materials usually have low imaginary indices that are not experimentally measurable and introducing large discrepancies with the theory. We tackled the k issue by adjusting the oxygen level within the last thin layer to fully control its absorption. We then qualify such dielectric stack resonance in TIRFM configuration for fluorescent viruses imaging.
Design in a proper way, optical dielectric resonant multilayers can support huge optical field enhancement when working under total internal reflection. TIRFM - Total Internal Reflection Fluorescence Microscopy, also based on total internal reflection illumination, is used in cell biology imaging where many biological processes involve cell membranes and their immediate intracellular spatial environment (signaling, cellular traffic, adhesion...). We propose to develop an enhanced version of TIRFM by investigating the optimization, conception and implementation of dedicated optical dielectric resonant multilayers as the glass coverslip replacement. Model samples such as lipid bilayers with a known thickness will be first investigated but our ultimate goal is to image more complex biological processes such as viral budding, or molecular transport mechanisms such as exocytosis or endocytosis.
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