Anita Mouttou

Fabien Lemarchand

Manon Gourdelier

Cihan Koc

Antonin Moreau

Julien Lumeau

Delphine Muriaux

Aude L Lereu

Cyril Favard

Enhanced TIRF microscopy using resonant dielectrics multilayers

SARS-COV-2 VLPs (GFP)

HIV- 1

 1 Photo-physicsCollection efficiency

3 )

 3 Resonant dielectric multilayer Control of the absorption of the top layerNumerical optimization with weighted targets to adapt the experimental conditions: 1) Spectral model for resonances 2) Numerically force the convergence on structures with good angular tolerance 3) Field maximization at the free interface of the structure in contact with the biological environment. 4) k value of the top layer? Fluorescence of beads for the 3 stacks evidencing the impact of k over the fluorescence enhancement. A plateau is reached with k  10 -4 . Optimizing the transmission 1) Maximizing the field 2) Large angular acceptance Δθ TIRF = 15 mrad

Acknowledgments

The authors acknowledge the CNRS for financial supports through the MITI -Mission for Transversal and interdisciplinary Initiatives 2017 and the 80|PRIME interdisciplinary program 2019 and the French Agence Nationale de la recherche AAP 2021. A. Mouttou is a recipient of a CNRS Ph.D. grant.


Is agroforestry an effective way to diversify cropping systems to improve biodiversity and ecosystem services?

Damien Beillouin, Tamara Ben-Ari, Eric Malézieux, Verena Seufert, David

Makowski

To cite this version:

Damien Beillouin, Tamara Ben-Ari, Eric Malézieux, Verena Seufert, David Makowski. Is agroforestry an effective way to diversify cropping systems to improve biodiversity and ecosystem services?. 5. Congrès mondial d'agroforesterie : en transition vers un monde viable, Université Laval, Jul 2022, Quebec, Canada. hal-04219573


Yousra Toumi

Anita Mouttou

Fabien Lemarchand

Guillaume Demesy

Cihan Koc

Delphine Muriaux

Antonin Moreau

Julien Lumeau

Cyril Favard
email: cyril.favard@irim.cnrs.fr

Aude L Lereu
email: aude.lereu@fresnel.fr

Resonant multi-dielectric coverslip for enhanced

Keywords: TIRF Microscopy, Dielectric Multilayer, Field enhancement, Evanescent wave

teaching and research institutions in France or abroad, or from public or private research centers.

INTRODUCTION

Total Internal Reflection Fluorescence microscopy also called TIRF microscopy is a technique that uses evanescent wave illumination to selectively excite fluorophores in a thin layer of the sample close to the coverslip. TIRF microscopy is particularly useful for studying processes that occur at the cell membrane. This technique, as its name suggests, is based on total reflection which occurs when the incident angle is greater than the critical angle. However, TIRF microscopy suffers from various limitations which can affect the optical contrast and the lateral resolution of the obtained biological images. For example, the problem of uneven illumination, due to the beam divergence, can degrade the image quality. In fact, the evanescent wave at the interface between the coverslip and the biological sample reaches its maximum intensity when the sample is illuminated at the critical angle. Yet, the exciting beam is inherently divergent thus a part of the beam is totally reflected which leads to the generation of the evanescent beam and another part, i.e the rays coming at angles less than the critical angle, propagates through the sample. To overcome this issue, we could increase the incident angle to guarantee a pure evanescent illumination of the sample. Unfortunately, this leads to a decay of the intensity of the evanescent field which degrades again the image quality. Dielectric thin films suggest promising prospects for amplifying the sensitivity of TIRF imaging due to their extreme confinement field property at the free interface when properly designed. Thin films are thin layers of material with a thickness typically ranging from a few nanometers to a few micrometers. They are used to modify the behavior of light, by reflecting, transmitting, or absorbing it in specific ways. Hence dielectric multilayers (DM) can be used to enhance locally the evanescent wave without introducing any further modifications into the optical arrangement of a traditional TIRF microscope. We showed that DM can be designed to support a desired field enhancement regardless of the excitation wavelength and incident angle [START_REF] Ndiaye | Optimal design for 100% absorption and maximum field enhancement in thin-film multilayers at resonances under total reflection[END_REF], [START_REF] Lereu | Multi-dielectric stacks as a platform for giant optical field[END_REF], [START_REF] Lereu | Scattering losses in multidielectric structures designed for giant optical field enhancement[END_REF] and with a defined enhancement at the free interface; here the biological medium. In the context of TIRF microscopy, we demonstrated the critical role of the angular divergence over the resulting fluorescence enhancement and the impact of the DM transmission over the spectral and angular range of the fluorescence collection [START_REF] Mouttou | Resonant dielectric multilayer with controlled absorption for enhanced total internal reflection fluorescence microscopy[END_REF]. Here we discuss a last parameter to be taken into account in the optimization of the DM to have a good theory-experiment agreement when measuring the fluorescence from beads [START_REF] Mouttou | Optimization of resonant dielectric multilayer for enhanced fluorescence imaging[END_REF].

RESULTS AND DISCUSSIONS

In this work, the DM stack consists of alternating quarter wave layers of SiO2, the low refractive index material, and Nb2O5, the high refractive index material. The stack ends with a layer of SiO2 to guarantee the biocompatibility. A stack is optimized to be resonant at an excitation wavelength of 561 nm and at an incident angle of 68° by adjusting the number of layers and the various thicknesses. Furthermore, the last layer of SiO 2 was replaced by a layer of SiOi, i < 2 to control the absorption and therefore adapt the field enhancement and the resonance angular tolerance. In fact, the extinction coefficient k of the top layer is a key parameter to control the field enhancement. However, classically it is difficult to experimentally determined values of k < 10 -4 -10 -5 , which is the typical value of dielectric thin film. Therefore, to fully control the optimization and obtain a good agreement between the predicted and experimentally measured fluorescence enhancement, we choose to use a material with a measurable k for the top layer of the DM. In order to preserve the bio compatibility of SiO2, we used SiOi, i < 2 a derivative of SiO2 by acting on the oxidization level of the Si target during deposition. We have realized and characterized by spectrophotometry a monolayer of SiOx. The index of this layer was measured to be ñ (561 nm) = 1.602+ 3.2 10 𝑖 -3 . The DM structure was optimized to be resonant at the same wavelength and incident angle as the initial stack. Therefore the physical thicknesses of the different layers of the stack were adjusted to be optimal at the operating point fixed by the biological application as shown in Fig. 1(a). A numerical evaluation was then done to study the effect of a variation of the k value of the SiOi layer on the fluorescence signal. The fluorescence enhancement factor was estimated for different values of k for the top layer, ranging from 10 -5 to 10 -2 while keeping the same physical thickness. An increase in the fluorescence enhancement was expected while decreasing k until than it reaches a plateau for k < 10 -4 . Experimentallly, three DM coated coverslips were made for this study; one ending with a layer of SiO2, one with a layer of SiOx and one with SiOy with x < y < 2. The fluorescence enhancement factor was introduced in a previous work [START_REF] Mouttou | Resonant dielectric multilayer with controlled absorption for enhanced total internal reflection fluorescence microscopy[END_REF] as the product of the exciation enhancement and the DM average transmission over the spectral and angular range of the fluorescence 𝑇𝑎𝑣 collection. The excitation enhancement, named , is defined by the ratio of the intensity measured at the DM-coated coverslip free interface to the one measured at the glass coverslip free interface, at the resonance angle and for a beam divergence . The expected fluorescence enhancement factor was then calculated as a function of incident angle for the three considered DM and for a classical glass coverslip as reference, see in Fig. 1(b). The maxima were obtained at the expected angular positions. The fluorescence enhancements were observed to be ×2.91, ×3.45 and ×4.86 with respect to the glass coverslip, for DM (SiOx), DM (SiOy) and DM (SiO2), respectively. Together with the field enhancement variation, a change in the angular tolerance for the three considered DM resonance was also observed. In fact, when the field enhancement increases, the full width at half maximum (FWHM) of the resonance decreases, in the presented cases, from 0.16° for DM (SiOx), to 0.13° for DM (SiOy) and even 0.038° for DM (SiO2). Or, for the fluorescence enhancement in an objective-based TIRF microscope, we need to find the best compromise between large field enhancement and angular tolerance to achieve the best fluorescence enhancement.

CONCLUSION

In conclusion, resonant dielectric multilayer stacks were designed to enhance the evanescent field at the excitation with an angular tolerance in accordance with the angular divergence fixed by the commercial TIRF- M. The DM transmission efficiency, over the angular and spectral range of the fluorescence collection, was also considered to compare the expected fluorescence with the measured one. This leads to improving the sensitivity of TIRF Microscopy by a factor of 4 for an angular divergence of 10 mrad. Here we will present the numerical predictions and the associated experiment in TIRF microscopy over fluorescence beads.

Figure 1 .

 1 Figure 1. (a): DM(SiOx) stack with an absorbing top layer of SiOx of extinction coefficient k=3.2 10 -3 (b): The angular distribution of the fluorescence enhancement factor, for the three samples (blue curves) compared to classical glass coverslip (gray curve)[START_REF] Lereu | Multi-dielectric stacks as a platform for giant optical field[END_REF].

ACKNOWLEDGEMENTS

The authors acknowledge the CNRS for financial supports through the 80|PRIME interdisciplinary program and the French research agency through the ANR NIS.


Yousra Toumi

Anita Mouttou

Fabien Lemarchand

Guillaume Demesy

Cihan Koc

Delphine Muriaux

Antonin Moreau

Julien Lumeau

Cyril Favard
email: cyril.favard@irim.cnrs.fr

Aude L Lereu
email: aude.lereu@fresnel.fr

Resonant multi-dielectric coverslip for enhanced total

Keywords: TIRF Microscopy, Thin films, Field enhancement, Evanescent beam

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

Total Internal Reflection Fluorescence microscopy also called TIRF microscpy is a technique that uses evanescent wave illumination to selectively excite fluorophores in a thin layer of the sample close to the cover slip. TIRF microscopy is particularly useful for studying processes that occur at the cell membrane. This technique, as its name suggests, is based on total reflection which occurs when the incident beam arrives at an angle greater than the critical angle so that it is mainly reflected when it encounters the interface between the cover slip and the biological sample. TIRF microscopy suffers from various limitations which can affect the optical contrast and the resolution of the obtained biological images. For example, the problem of uneven illumination, due to the beam divergence, can degrade the image quality. In fact, the evanescent beam reaches its high intensity when the sample is illuminated at the critical angle. Yet, the exciting beam is inherently divergent thus a part of the beam is totally reflected which leads to the essence of the evanescent beam and another part, i.e the rays coming at angles less then the critical angle, propagates through the sample. To overcome this issue, we can increase the incident angle to guarantee an even illumination of the whole sample. Unfortunately, this leads to a decrease of the evanescent beam's intensity which degrades again the image quality. Thin films suggest promising prospects for amplifying the sensitivity of TIRF imaging due to their extreme confinement field property at the interface. Thin films are thin layers of material with a thickness typically ranging from a few nanometers to a few micrometers. They are used to modify the behavior of light, by reflecting, transmitting, or absorbing it in specific ways. Hence dielectric multilayers can be used ,due to their bio-compatibility property, to enhance locally the evanescent beam without introducing any modifications into the traditional TIRF microscope.

In this work, the multilayer stack consists of alternating quarter wave layers of SiO2 , the low refractive index material, and Nb2O5, the high refractive index material. The stack ends with an absorbing layer of SiOx. We actually replaced the last layer SiO2 by SiOx to control the absorption. In fact, one major parameter used to control the exaltation of the field is the extinction coefficient k which is also the imaginary part of the refractive index of a given material. We can't determine experimentally values of k < 10 -3 and thus we can't use SiO2 as absorbing layer and control k (ref k(SiO2) = 10 -5 ). We shall use a material with a measurable k. We decided hence to modify chemically the last layer in order to preserve the bio compatibility property. We controlled the oxidization level of the ending layer while deposit to transition from SiO2 to SiOx. The refractive index of this layer is then determined by spectrophotometry which leads to a value of n = 1.602 + i0.0032 at 561nm. the exaltation is 5 times less important but its full width at half maximum is ten times greater. insert graphs

Conclusion

In conclusion, resonant dielectric multi-layer stacks were designed to enhance the evanescent beam which leads to improving the sensitivity of TIRF Microscopy. We carried out numerical simulations to predict the optical response of the designed component. The increase of the value of the extinction coefficient of the top layer leads to a drop of the enhancement at the free interface but helps increase the angular tolerance at the resonance.

Acknowledgements

The authors acknowledge the CNRS for financial supports through the 80-PRIME interdisciplinary program and the French research agency through the ANR NIS.