SARS-CoV-2 is an RNA enveloped virus responsible for the COVID-19 pandemic that conducted in 6 million deaths worldwide so far. SARS-CoV-2 particles are mainly composed of the 4 main structural proteins M, N, E and S to form 100 nm diameter viral particles. Based on productive assays, we propose an optimal transfected plasmid ratio mimicking the viral RNA ratio in infected cells. This allows SARS-CoV-2 Virus-Like Particle (VLPs) formation composed of the viral structural proteins M, N, E and mature S. Furthermore, fluorescent or photoconvertible VLPs were generated by adding a fluorescent protein tag on N or M mixing with unlabeled viral proteins and characterized by western blots, atomic force microscopy coupled to fluorescence and immuno-spotting. Thanks to live fluorescence and super-resolution microscopies, we quantified VLPs size and concentration. SARS-CoV-2 VLPs present a diameter of 110 and 140 nm respectively for MNE-VLPs and MNES-VLPs with a concentration of 10e12 VLP/ml. In this condition, we were able to establish the incorporation of the Spike in the fluorescent VLPs. Finally, the Spike functionality was assessed by monitoring fluorescent MNES-VLPs docking and internalization in human pulmonary cells expressing or not the receptor hACE2. Results show a preferential maturation of S on N(GFP) labeled VLPs and an hACE2-dependent VLP internalization and a potential fusion in host cells. This work provides new insights on the use of non-fluorescent and fluorescent VLPs to study and visualize the SARS-CoV-2 viral life cycle in a safe environment (BSL-2 instead of BSL-3). Moreover, optimized SARS-CoV-2 VLP production can be further adapted to vaccine design strategies.

Total internal reflection fluorescence microscopy (TIRF-M) is widely used in biological imaging. Evanescent waves, generated at the glass-sample interface, theoretically strongly improve the axial resolution down to a hundred of nanometers. However, objective based TIRF-M suffers from different limitations such as interference fringes and uneven illumination, mixing both propagating and evanescent waves, which degrade the image quality. In principle, uneven illumination could be avoided by increasing the excitation angle, but this results in a drastic loss of excitation power. We designed dedicated 1D photonic crystals in order to circumvent this power loss by directly acting on the intensity of the evanescent field at controlled incident angles. In this framework, we used dedicated resonant multi-dielectric stacks, supporting Bloch surface waves and resulting in large field enhancement when illuminated under the conditions of total internal reflection. Here, we present a numerical optimization of such resonant stacks by adapting the resulting resonance to the angular illumination conditions in TIRF-M and to the fluorescence collection constraints. We thus propose a dedicated resonant structure with a control of the absorption during thin film deposition. A first experimental demonstration illustrates the concept with a 3-fold fluorescence enhancement in agreement with the numerical predictions.