e-Substrates for molecular imaging in cells – NIS

The NIS project is divided in 3 main work packages:
1- Numerical study for the design and optimization of the components (PAM bi-colore and NIS)
2- Fabrication and characterization of the components
3- Implementation of the substrates in TIRF-M for real time viral assemblies assay.

In 1, we pursue three different tasks, one is improving the optimization of the resonant planar all-dielectric multilayers (PAM) by taking into account the emission angular divergence and the collection efficienty. The second task is then to further push the concept of PAM by looking at multiple resonances. Intrinsically, PAM possess several resonances for the couple (incident angle, excitation wavelength). The optimization is then thought to obtain resonances for two distinct wavelengths but for the same incidence and possibly similar factors of enhancement. Finally, the third task is to numerically design the structured PAM, i.e. Nanophotonics improving substrate – NIS, to couple field enhancement and confinement with a spatial distribution of the local field down to lambda/3. The numerical optimization of the NIS will be carried out using appropriate (2D/3D) finite element formulations.

In 2, the components are fabricated combining the optical thin film deposition expertise within the Espace Photonique platform at Institut Fresnel and the expertise in RIE etching and lithography within the MIMENTO platform at FEMTO-ST. Optimal masking and etching procedures are being optimized. The resulting PAM/NIS are then characterized by optical spectroscopy and/or near field optical microscopy to evaluate their structural parameters and optical response.

In 3, we want to assess the substrates for microscopy in real biological conditions, including BSL3 facility microscopes at IRIM. Each new substrate will first be tested with fully characterized molecular systems such as supported lipid membranes, virus like particles which, size, composition and dynamics are known. Then, the specifically designed substrate will be implemented to the different fluorescence techniques commonly used in the IRIM . We will then use the 2-color PAM to simultaneously observe the dynamics of the main viral protein and cellular molecules (lipids or actin) using Imaging-FCS (Im-FCS). The local field enhancement will authorize a better-defined mapping of the dynamic properties of each molecule near the diffraction limit, which is much better than what is currently achieved. Finally, we will test NIS and investigate it to perform simultaneous multi-spots fluorescence correlation spectroscopy allowing hundreds of simultaneous measurements with ms to sub-ms temporal resolution within one cell at a lambda/6 lateral resolution.

In the context of TIRF microscopy, we have proposed to use stacks of dielectric thin films designed to support controllable field strengths at the free interface of the stacks under total reflection illumination, whatever the illumination conditions (wavelength, angle of incidence, polarisation). The aim is to improve the sensitivity of TIRF-M. The compatibility with living systems and the optically transparent nature of these stacks are assets for their immediate use in current TIRF-M techniques. We have already shown that with this type of component we can gain a factor of 4 in the detection of fluorescently labelled SARS-CoV-2 viruses deposited on the surface of the developed coverslip using a commercial TIRF microscope.

Using a dedicated TIRF microscope, we have demonstrated a record 50-fold increase in fluorescence. We are currently working with the company Abbelight to exploit these components.

Finally, we are currently taking the concept a step further by developing structured multilayer substrates that enable enhancement of the field while controlling its spatial confinement. While retaining the improvement in sensitivity already demonstrated, we hope to improve lateral resolution through nanostructured illumination resulting from a local interference pattern arising from the specific structuring of the stack. Initial prototypes have been made and are currently being characterized and implemented in TIRF-M.

The ultimate goal of the NIS project is to specifically design nanophotonic improving substrates to authorize nano-bio-imaging with basic optical microscopes. The major innovation and ingenuity of this project is to achieve SR-SIM coupled with SW-TIRF-M by only adding a biocompatible optimized substrate directly adaptable to TIRF microscopes. This innovation will benefit to the numerous biological-imaging platforms present in France and worldwide. The interdisciplinary nature of this project will not only lead to originality in biophotonics, but at least 4 different fields will benefit from original and ambitious developments.

In terms of optical resonance or field enhancement in PAM, our optimization method allows accessing field enhancement with at least one decade more than periodic dielectric structures. Furthermore, introducing micro- to nano-structuration pushes the NIS concept further than PAM where we aim in combining the best of both worlds: i.e. controlled field enhancement and spectral multi-functions with PAM with lateral confinement with the created standing wave resulting by the structuration.

In terms of fabrication challenges here is the capability to structure materials that can etch very differently, resulting in rough and undefined boundaries. The results of this project will pave a way to a wide range of optical components including antireflection coatings, pixelated filters or optical metasurfaces.

Finally, our approach will make it possible to acquire images with a high sensitivity and high spatial (lambda/10 on spatially structured PAM) and temporal (< 0.1ms) resolution. Using the proposed NIS to improve the fluorescent signal without increasing the laser excitation noise will also benefit to techniques such as multispot and imaging FCS or spt-PALM.

1- Mouttou, A., et al, Optimization of resonant dielectric multilayer for fluorescence imaging, Optical Materials : X 17, 100223 (2023).
2- Mouttou, A., et al, Resonant dielectric multilayer with controlled absorption for enhanced fluorescence imaging, Opt. Express 30, 15365 (2022).

3- Mouttou, A., Lumeau, J., Lereu, A. L., Favard, C. Lame optique biocompatible destine´e a` la microscopie a` re´flexion totale interne et syste`me d’imagerie microscopique comportant une telle lame Brevet FR 2108879 - 24/08/2021.
4- Mouttou, A., Lemarchand, F., Lumeau, J., Lereu, A. L., Favard, C. HARI-coverslips - High Amplitude Resonant Improved – Coverslip Brevet FR 2301673 - 23/02/2023.

Based on the use of evanescent waves, total reflection fluorescence microscopy (TIRF-M) allows observing events occurring at the interface between the microscope coverslip and the biological sample. In the cell, this interface corresponds to the plasma membrane, where many processes such as endo/exocytosis and cell adhesion; fundamental for cell survival; take place. It is also at the plasma membrane that many human pathogenic viruses (HIV, Sars-CoV2, Influenza) assemble and bud, or exit through it. TIRF microscopy is therefore widely used as the observation method of choice in many fields of membrane biology. If this technique is relatively simple to use, its sensitivity and lateral resolution remain limiting factors for the study of nanometer-sized objects such as viruses. In the NIS project, we propose to act on the local field excitation by introducing 1) an enhancement (i.e. for sensitivity) and/or 2) an interference pattern (i.e. for lateral resolution) of the evanescent field at the microscope coverslip free interface. The originality of the project lies in the design (WP1), fabrication (WP2) and validation (WP3) of enhancing and/or patterning substrates at the scale of the conventional microscope coverslip and thus directly adaptable to all TIRF microscopes.

The starting structures chosen to meet these objectives are planar multi-dielectric stacks (PAMs) which can be designed to withstand field strengths up to 10^4 under any illumination conditions. This makes them very attractive for improving sensitivity in TIRF microscopy. However, PAMs are originally designed for plane wave illumination which does not correspond to the illumination conditions in microscopy. In this project, we want to push this concept further and adapt it to TIRF microscopy by taking into account the illumination conditions by the objective (thus with high angular divergence) as well as the fluorescence collection by the same objective (WP1-T1 and WP2-T1).

Meanwhile, we also want to be able to image two molecules simultaneously to either visualize the molecular interconnection between the cell and the virus during viral assembly, or to observe in real time the assembly of different viral proteins. In NIS project, we will use HIV-1 assembly in infected living T cells as a model (WP3-T2). For this purpose, we will adapt the design and implementation of the PAM for the development of a two-colors PAM with the same order of magnitude of enhancement factor and with close incident angles (WP1-T2 and WP2-T1).

Finally, we propose to extend our concept to improve both the sensitivity in TIRF-M and its lateral resolution thanks to an enhanced structured illumination with an estimated spatial resolution of ?/10 (already designed numerically WP1-T2 and T3). In this case, we will structure the PAM by optimizing the trade-off between the large field enhancement given by the PAM and the field distribution introduced by the 3D nano- micro-structuration of the PAM; resulting in what we call Nanophotonics Improving Substrate (NIS). The final goal of our project is to validate their use to i) improve the accuracy in single molecule microscopy and the sensitivity in correlation imaging to follow the dynamics of molecular events occurring at the cell membrane, such as viral budding, ii) perform fast and sensitive imaging with a sub-wavelength spatial resolution.


The microscope coverslips designed here will be quickly and easily marketed as a microscopy consumable accessible to the biology community while allowing a clear gain in the sensitivity and lateral resolution of the observed sample.