NIR lanthanide based bioprobes for Two Photon Scanning Laser Microscopy

L’objectif de cette thèse est l’élaboration de sondes à base de lanthanide pour la microscopie optique biphotonique. Cette technique d’imagerie complémentaire à l’IRM et au scanner permet une analyse rapide et facile de tissus épais. Afin de permettre l’observation en profondeur, l’absorption et l’émission de la sonde doit se situer dans la zone de transparence biologique [700 – 1200 nm]. L’absorption à deux photons (ADP) est un phénomène d’optique linéaire de troisième ordre par lequel l’état excité est atteint par absorption simultanée de deux photons. De fait, l’excitation à énergie moitié se situe dans la zone de transparence biologique. Les sondes envisagées combineraient les propriétés optiques uniques des lanthanides, telles que des bandes d’émission très étroites allant du visible à l’infrarouge et des durées de vie de luminescence longues, et les avantages de l’ADP, permettant une excitation dans l’IR et une résolution tridimensionnelle. Dans ce contexte, cette thèse décrit l’élaboration de complexes d’europium et d’ytterbium à ligands macrocycliques stables en milieu aqueux et dont la luminescence peut être sensibilisée à deux photons. Ces complexes ont permis l’imagerie de la vascularisation de cerveaux de souris par microscopie biphotonique dans le proche infrarouge. La seconde approche consiste à encapsuler un complexe luminescent dans des nanoparticules desilice formées par la technique sol-gel (collaboration A. Ibanez, institut Néel, Grenoble) afin de protéger le complexe du milieu biologique. Enfin la dernière approche consiste à greffer des complexes de lanthanides à la surface d’une particule de silice par chimie organométallique de surface. Ces travaux ont conduit à la formation de nano-objet très brillants dans le rouge et le proche infrarouge, détectables à l’échelle de l’objet unique par microscopie à deux photons.Two Photon Scanning Laser Microscopy (TPSLM) has evolved as an emerging bio-imaging technique widely used in academic research and in medical diagnosis. This technique requires the design of bioprobes specially optimized for such purpose. A particular attention is actually devoted bio-probes featuring both two-photon absorption (TPA) and emission in the near infra-red (NIR) spectral range [700 – 1200 nm], also called biological window that is particularly promising for thick tissues imaging. In this context, europium complexes emitting in the red (615 nm) has been recently sensitized by two photon antenna effect and used for TPSLM in cells combining the advantages of lanthanide emission (sharp line and long lived) and those of TPA. Based on this preliminary results, this thesis describe the design of europium and ytterbium complexes which have an improved stability in water and good emission properties sensitized by TPA. Theses complexes allow the imaging of mice’s brain vascularisation in the NIR. An another approach to stabilize lanthanide complexes was also used by encapsulating theses fluorophores in silica nanoparticle (collaboration with A. Ibanez, Institut Néel, Grenoble). Then the last approach consists on the grafting of the chromophores on silica sphere using surface organometallic chemistry methods. The nanoparticles obtained by both way are really luminescent in the red or infrared and can be imaged as single nanoparticle by TPSLM

Conception de sondes et nano-sondes à base de lanthanides émettant dans le proche infrarouge pour la microscopie biphotonique

L objectif de cette thèse est l élaboration de sondes à base de lanthanide pour la microscopie optique biphotonique. Cette technique d imagerie complémentaire à l IRM et au scanner permet une analyse rapide et facile de tissus épais. Afin de permettre l observation en profondeur, l absorption et l émission de la sonde doit se situer dans la zone de transparence biologique [700 1200 nm]. L absorption à deux photons (ADP) est un phénomène d optique linéaire de troisième ordre par lequel l état excité est atteint par absorption simultanée de deux photons. De fait, l excitation à énergie moitié se situe dans la zone de transparence biologique. Les sondes envisagées combineraient les propriétés optiques uniques des lanthanides, telles que des bandes d émission très étroites allant du visible à l infrarouge et des durées de vie de luminescence longues, et les avantages de l ADP, permettant une excitation dans l IR et une résolution tridimensionnelle. Dans ce contexte, cette thèse décrit l élaboration de complexes d europium et d ytterbium à ligands macrocycliques stables en milieu aqueux et dont la luminescence peut être sensibilisée à deux photons. Ces complexes ont permis l imagerie de la vascularisation de cerveaux de souris par microscopie biphotonique dans le proche infrarouge. La seconde approche consiste à encapsuler un complexe luminescent dans des nanoparticules desilice formées par la technique sol-gel (collaboration A. Ibanez, institut Néel, Grenoble) afin de protéger le complexe du milieu biologique. Enfin la dernière approche consiste à greffer des complexes de lanthanides à la surface d une particule de silice par chimie organométallique de surface. Ces travaux ont conduit à la formation de nano-objet très brillants dans le rouge et le proche infrarouge, détectables à l échelle de l objet unique par microscopie à deux photons.Two Photon Scanning Laser Microscopy (TPSLM) has evolved as an emerging bio-imaging technique widely used in academic research and in medical diagnosis. This technique requires the design of bioprobes specially optimized for such purpose. A particular attention is actually devoted bio-probes featuring both two-photon absorption (TPA) and emission in the near infra-red (NIR) spectral range [700 1200 nm], also called biological window that is particularly promising for thick tissues imaging. In this context, europium complexes emitting in the red (615 nm) has been recently sensitized by two photon antenna effect and used for TPSLM in cells combining the advantages of lanthanide emission (sharp line and long lived) and those of TPA. Based on this preliminary results, this thesis describe the design of europium and ytterbium complexes which have an improved stability in water and good emission properties sensitized by TPA. Theses complexes allow the imaging of mice s brain vascularisation in the NIR. An another approach to stabilize lanthanide complexes was also used by encapsulating theses fluorophores in silica nanoparticle (collaboration with A. Ibanez, Institut Néel, Grenoble). Then the last approach consists on the grafting of the chromophores on silica sphere using surface organometallic chemistry methods. The nanoparticles obtained by both way are really luminescent in the red or infrared and can be imaged as single nanoparticle by TPSLM.LYON-ENS Sciences (693872304) / SudocSudocFranceF

d-f heterobimetallic association between ytterbium and ruthenium carbon-rich complexes: redox commutation of near-IR luminescence.

International audienceWe describe how the association between an ytterbium ion and a ruthenium carbon-rich complex enables the first switching of the near-IR Yb(III) luminescence by taking advantage of the redox commutation of the carbon-rich antenna

Core-shell and doped silicate nanoparticles prepared by a spray-drying process for the development of biological luminescent tracer

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Doped silica nanoparticles containing two-photon luminescent Eu(III) complexes for the development of water stable bio-labels

International audienceFunctionalized tris-dipicolinate europium(III) complexes present a very important two-photon absorption cross-section (up to 775 GM) but exhibit a weak stability in aqueous solutions due to the ligand substitution by water molecules. This dissociation decreases radically the luminescence quantum yield and lifetime of the complex. In this paper, we present a one-step alternative route to stabilize these lanthanide complexes in aqueous solutions or in aqueous buffer solutions by embedding them in silica nanoparticles (NPs). This simple one-step method is based on the spray-drying of sol-gel solutions involving silicon alkoxides as precursors of the silica NPs, hydrolyzed through a small amount of water, an organic solvent and the dissolved Eu(III) complex. These atomized sols are dried under laminar air flow in a tubular furnace while the resulting NPs were collected with an electrostatic filter. Different types of silicon alkoxides were used to adjust the hydrophilic character and porosity ofNPs, to optimize their biocompatibility and dispersion in aqueous solutions and to avoid the Eu(III) complexes dissociation. The chemical stability in aqueous solutions of the Eu-complexes inserted in the different NPs was followed through their luminescence spectroscopy and two-photon microscopy to determine the optimal chemical composition of the starting sols for the preparation of highly luminescent water stable bio-labels

In solution sensitization of Er(III) luminescence by the 4-tetrathiafulvalene-2,6-pyridinedicarboxylic acid dimethyl antenna ligand

International audienceIn the [Er(hfac)(3)(L)](2) complex (1) (L = 4-tetrathiafulvalene-2,6-pyridinecarboxylic acid dimethyl ester), the Er(III) ion is bonded to the tridentate coordination site. Electrochemical and photophysical measurements in solution reveal that the tetrathiafulvalene moiety is a versatile antenna for erbium luminescence sensitization at 6540 cm(-1) upon excitation in the low-energy charge transfer transition (donor to acceptor charge transfer) at 16600 cm(-1) assigned via time-dependent density functional theory calculations

Bright Luminescent Silica Nanoparticles for Two-Photon Microscopy Imaging via Controlled Formation of 4,4′-Diethylaminostyryl-2,2′-bipyridine Zn(II) Surface Complexes

International audienceSilica nanoparticles with a surface functionalized by two-photon absorbing (≡SiO)Zn(OSi(OtBu)3)(DEAS-bipy) chromophores are prepared via surface chemistry controlled at the molecular level. This involves the grafting of {Zn[OSi(OtBu)3]2}2 on the surface silanols of a silica partially dehydroxylated at 700 °C followed by the coordination of DEAS-bipy. The spectroscopic and photophysical properties of the grafted species (≡SiO)Zn(OSi(OtBu)3)(DEAS-bipy) compare well with that of the molecular model {DEAS-bipyZn(OAc)2} with the advantage of allowing a high density of chromophores on a nanometric object (ca. 200 chromophores per silica particle of 12 nm). These particles are luminescent and exhibit a giant two-photon cross-section of about 90 × 103 GM; such two-photon brightness allows the imaging of a single nanoparticle using two-photon scanning microscopy

Influence of the Metal Ion on the Two-Photon Absorption Properties of Lanthanide Complexes Including Near-IR Emitters.

International audience: The synthesis of tris(2-thenoyltrifluoroacetonate)lanthanide(III) complexes featuring a diethylaminostyryl-2,2'-bipyridine coligand was achieved for lanthanum; the near-infrared (NIR) emitters neodymium, erbium, and ytterbium; and the transition-metal yttrium. The photophysical properties were thoroughly studied, and it was demonstrated that the conjugated bipyridine ligand acts as a good antenna for the sensitization of the NIR emitters. The two-photon absorption (TPA) properties of all five complexes were investigated by using both two-photon excited fluorescence and the Z-scan method. We demonstrate that the nature of the rare earth ion has almost no influence on the TPA properties centered on the conjugated bipyridyl ligand. Finally, we show that Yb(III) is sensitized by a two-photon antenna effect, and that Nd(III) is mostly sensitized by a one-photon process involving direct excitation of forbidden f-f transitions