Ecole Doctorale

Physique et Sciences de la Matière

Spécialité

PHYSIQUE & SCIENCES DE LA MATIERE - Spécialité : OPTIQUE, PHOTONIQUE ET TRAITEMENT D'IMAGE

Etablissement

Aix-Marseille Université

Mots Clés

FRET,nanophotonique,biophotonique,,

Keywords

FRET,nanophotonics,biophotonics,,

Titre de thèse

Transfert d'énergie de Förster exalté par des nanoantennes optiques
Enhancing Förster resonance energy transfer at longer distances using metal nanostructures.

Date

Thursday 23 September 2021

Adresse

52 Avenue Escadrille Normandie Niemen, 13013 Amphi Rouard

Jury

Directeur de these M. Jérôme WENGER Institut Fresnel
Examinateur Mme María GARCíA-PARAJO ICFO Barceolna
Examinateur M. Emmanuel MARGEAT CBS Montpellier
Examinateur M. Manos MAVRAKIS Institut Fresnel
Rapporteur M. Mathieu MIVELLE Institut des NanoSciences de Paris - UPMC
Rapporteur M. Guillermo ACUñA University of Fribourg. Department of Physics

Résumé de la thèse

Le transfert d’énergie de Förster est une technique de microscopie permettant l’étude de la structure de biomolécules et des interactions entre molécules isolées. Cette technique est définie comme une résultante de l’interaction dipole-dipole entre deux molécules. Le FRET étant une technique précise lorsque la distance intermoléculaire est inférieure à 10 nanomètres, l’objectif de cette thèse était de développer le FRET sur des distances entre molécules supérieures à cette limite. En effet, l’efficacité de FRET est le résultat de l’interaction entre les plasmons de surfaces des atomes métalliques et les molécules fluorescentes. Dans ce cadre, plusieurs systèmes biologiques ont été étudiés dans des nanostructures plasmoniques pour mesurer le transfert d’énergie de Förster.

Thesis resume

Single molecule Förster resonance energy transfer (smFRET) is a major technique to measure biomolecular conformations and interactions. However, its range is limited to nanometer distances and nanomolar concentrations. The objective of this PhD thesis is to extend the applicability of smFRET using optical nanostructures such as aluminum nanoapertures to achieve long distance, biologically-relevant micromolar concentration and improved temporal resolution. This breakthrough is achieved by introducing novel nanophotonic elements to manipulate energy transfer at the nanoscale and go significantly beyond the conventional diffraction-limited microscopes. The thesis outcomes will benefit many applications in structural biology, drug discovery and energy conversion at the nanoscale. In order to improve the single-molecule sensitivity of FRET measurements at longer distances, the classical FRET excitation scheme is improved using pulsed-interleaved excitation (PIE) of two molecules independently with 12.5 ns delay, i.e. one is using the green and red excitation lines at 557 and 633 nm, respectively. Therefore, the FRET pairs lacking donor and acceptor fluorophore, are excluded from post-analysis and the peak of the FRET efficiency which is close to zero is no longer present on the PIE-FRET histogram. Taking advantage of the interaction between surface plasmons of metal and fluorescent molecules, one can achieve the significant enhancement of fluorescence compared to diffraction-limited confocal microscope. Hence, the interaction between fluorophores can be mediated by metal nanostructure. Reducing drastically the observation volume down to zeptoliter volume (10-21 L), the single-molecule observations can be achieved with micromolar concentrations which is highly important for studying the interactions between macromolecules. Using two FRET pairs of the dyes possessing quite different chemical structure, we detect FRET interaction at the distance of 13.6 nm. We probe different diameters of nanoapertures in order to obtain the higher fluorescence gain and get the narrower FRET distributions. We elaborate the passivation protocol of metal surface to prevent any non-specific interaction between surface and the molecules. We obtain the lifetime information about different cell compartments (actin, septin and plasma membrane) by time-correlated single-photon measurements (TCSPC) to study the interactions at intracellular level. The interaction between free electrons of metal and fluorophore can modify the intrinsic properties of the dye and therefore its lifetime. Lifetime change can be related the distance information of a dye from metal surface. The interaction between the dye and electrons of metal occurs at the range far beyond 10 nm which extends the scope of energy transfer study for the thesis. The exploiting of nanoapertures opens the possibility for measuring macromolecular interactions. We study the association between antitermination protein LicT and RNA hairpin by means of 2-color Fluorescence Cross-Correlation Spectroscopy (FCCS). The main goal is to determine the binding fraction of LicT protein to RNA and binding constant defining the interaction. We access the conformational dynamics by PIE-FRET for mGluR protein upon the addition of glutamate receptor which is the major excitatory neurotransmitter in the central nervous system.