Comparison of the photoluminescence properties of semiconductor quantum dots and non-blinking diamond nanoparticles. Observation of the diffusion of diamond nanoparticles in living cells

Long-term observations of photoluminescence at the single-molecule level were until recently very diffcult, due to the photobleaching of organic ?uorophore molecules. Although inorganic semiconductor nanocrystals can overcome this diffculty showing very low photobleaching yield, they suffer from photoblinking. A new marker has been recently introduced, relying on diamond nanoparticles containing photoluminescent color centers. In this work we compare the photoluminescence of single quantum dots (QDs) to the one of nanodiamonds containing a single-color center. Contrary to other markers, photoluminescent nanodiamonds present a perfect photostability and no photoblinking. At saturation of their excitation, nanodiamonds photoluminescence intensity is only three times smaller than the one of QDs. Moreover, the bright and stable photoluminescence of nanodiamonds allows wide field observations of single nanoparticles motion. We demonstrate the possibility of recording the tra jectory of such single particle in culture cells

25-nm diamond crystals hosting single NV color centers sorted by photon-correlation near-field microscopy

Diamond nanocrystals containing highly photoluminescent color centers are attractive non-classical and near-field light sources. For near-field applications the size of the nanocrystal is crucial since it defines the optical resolution. NV (Nitrogen-Vacancy) color centers are efficiently created by proton irradiation and annealing of a nanodiamond powder. Using near-field microscopy and photon statistics measurements, we show that nanodiamond with size down to 25 nm can hold a single NV color center with bright and stable photoluminescence

BRET and Time-resolved FRET strategy to study GPCR oligomerization: from cell lines toward native tissues

The concept of oligomerization of G protein-coupled receptor (GPCR) opens new perspectives regarding physiological function regulation. The capacity of one GPCR to modify its binding and coupling properties by interacting with a second one can be at the origin of regulations unsuspected two decades ago. Although the concept is interesting, its validation at a physiological level is challenging and probably explains why receptor oligomerization is still controversial. Demonstrating direct interactions between two proteins is not trivial since few techniques present a spatial resolution allowing this precision. Resonance energy transfer (RET) strategies are actually the most convenient ones. During the last two decades, bioluminescent resonance energy transfer and time-resolved fluorescence resonance energy transfer (TR-FRET) have been widely used since they exhibit high signal-to-noise ratio. Most of the experiments based on GPCR labeling have been performed in cell lines and it has been shown that all GPCRs have the propensity to form homo- or hetero-oligomers. However, whether these data can be extrapolated to GPCRs expressed in native tissues and explain receptor functioning in real life, remains an open question. Native tissues impose different constraints since GPCR sequences cannot be modified. Recently, a fluorescent ligand-based GPCR labeling strategy combined to a TR-FRET approach has been successfully used to prove the existence of GPCR oligomerization in native tissues. Although the RET-based strategies are generally quite simple to implement, precautions have to be taken before concluding to the absence or the existence of specific interactions between receptors. For example, one should exclude the possibility of collision of receptors diffusing throughout the membrane leading to a specific FRET signal. The advantages and the limits of different approaches will be reviewed and the consequent perspectives discussed

Fluorescent ligands to investigate GPCR binding properties and oligomerization

Abstract Fluorescent ligands for GPCRs (G-protein-coupled receptors) have been synthesized for a long time but their use was usually restricted to receptor localization in the cell by fluorescent imaging microscopy. During the last two decades, the emergence of new fluorescence-based strategies and the concomitant development of fluorescent measurement apparatus have dramatically widened the use of fluorescent ligands. Among the various strategies, TR (time-resolved)-FRET (fluorescence resonance energy transfer) approaches exhibit an interesting potential to study GPCR interactions with various partners. We have derived various sets of ligands that target different GPCRs with fluorophores, which are compatible with TR-FRET strategies. Fluorescent ligands labelled either with a fluorescent donor (such as europium or terbium cryptate) or with a fluorescent acceptor (such as fluorescein, dy647 or Alexa Fluor ® 647), for example, kept high affinities for their cognate receptors. These ligands turn out to be interesting tools to develop FRET-based binding assays. We also used these fluorescent ligands to analyse GPCR oligomerization by measuring FRET between ligands bound to receptor dimers. In contrast with FRET strategies, on the basis of receptor labelling, the ligand-based approach we developed is fully compatible with the study of wild-type receptors and therefore with receptors expressed in native tissues. Therefore, by using fluorescent analogues of oxytocin, we demonstrated the existence of oxytocin receptor dimers in the mammary gland of lactating rats

Characterization of the livestock production system and potential for enhancing productivity through improved feeding in Amoni Division, Mweiga District, Central Kenya, May 2010

Lipid droplets (LDs) are intracellular lipid-rich organelles that regulate the storage of neutral lipids and were recently found to be involved in many physiological processes, metabolic disorders, and diseases including obesity, diabetes, and cancers. Herein we present a family of new fluorogenic merocyanine fluorophores based on an indolenine moiety and a dioxaborine barbiturate derivative. These so-called StatoMerocyanines (SMCy) fluoresce from yellow to the near-infrared (NIR) in oil with an impressive fluorescence enhancement compared to aqueous media. Additionally, SMCy display remarkably high molar extinction coefficients (up to 390 000 M^–1 cm^–1) and high quantum yield values (up to 100%). All the members of this new family specifically stain the LDs in live cells with very low background noise. Unlike Nile Red, a well-known lipid droplet marker, SMCy dyes possess narrow absorption and emission bands in the visible, thus allowing multicolor imaging. SMCy proved to be compatible with fixation and led to high-quality 3D images of lipid droplets in cells and tissues. Their high brightness allowed efficient tissue imaging of adipocytes and circulating LDs. Moreover their remarkably high two-photon absorption cross-section, especially SMCy5.5 (up to 13 300 GM), as well as their capacity to efficiently fluoresce in the NIR region led to two-photon multicolor tissue imaging (liver). Taking advantage of the available color palette, lipid droplet exchange between cells was tracked and imaged, thus demonstrating intercellular communication

Nanoparticules de diamant fluorescentes comme marqueurs pour les cellules: étude de leurs propriétés optiques et de leur mécanisme d'internalisation

This thesis work studies the use of Photoluminescent NanoDiamonds (PNDs) for bio-imaging applications. Nanodiamonds are photoluminescent thanks to embedded nitrogen-vacancy (NV) color centers. The thesis is divided in two parts. The first part concerns the study of the optical properties of NV color centers in nanodiamonds. After optimization of the NV center concentration, we compared the photoluminescence of PNDs to commercial Quantum Dots (QDs) and conclude that it can be similar or even higher in the case of PNDs. To enhance the imaging contrast of internalized by cells PNDs, we studied the 2-photon excitation properties of NV centers. While implementing a pulsed excitation laser, we discovered that simultaneous one- and two-photon excitation (IR+VIS pulses) quenches the photoluminescence signal of PNDs. We examined how this effect can serve for super-resolution imaging of NV color centers in nanodiamonds. The second part of the work is devoted to the applications of PNDs as bio-imaging probes. In the prospect of applications of PNDs as drug delivery vehicles, we studied the uptake mechanisms of PNDs and elucidated their intracellular localization by blocking different entry mechanisms and by immunofluorescence experiments. Moreover, we ensured that PNDs are not toxic for cells in culture. As a first try of vectorization we covered PNDs with plasmid DNA and examined the transfection efficiency.Ce travail de thèse porte sur l'utilisation des NanoDiamants Photoluminescents (NDPs) pour des applications en bio-imagerie. Les nanodiamants (NDs) sont photoluminescents grâce à la présence de centres colorés azote-lacune (NV) dans leur maille cristalline. Le manuscrit est divisé en deux parties. La première concerne l'étude des propriétés optiques des centres colorés NV dans des NDs. Après l'optimisation de la concentration des centres NV, nous comparons la photoluminescence des NDPs à celle des nanoparticules semi-conductrices commerciales; nous concluons qu'elle peut être équivalente, même supérieure dans le cas des NDPs. Pour augmenter le contraste d'imagerie intracellulaire des NDPs, nous avons étudié l'excitation à 2-photons des centres NV. Lors de cette étude avec un laser impulsionnel, nous avons découvert que le signal de photoluminescence des NDPs excité à un photon chute très fortement lorsque l'impulsion infrarouge est simultanée de l'excitation visible. Nous avons étudié la façon d'utiliser cet effet pour l'imagerie de super-resolution. La deuxième partie porte sur l'étude des applications des NDPs comme sondes pour la bio-imagerie. Dans le but d'utiliser des NDPs comme véhicules de biomolécules, nous avons étudié leurs mécanismes d'internalisation et avons élucidé leur localisation intracellulaire, en inhibant des voies différentes d'internalisation et par des expériences d'immunofluorescence. De plus, nous avons montré que les NDPs ne sont pas toxiques pour des cellules en culture. Un premier essai de vectorisation a été mené avec de NDPs couverts d'ADN plasmidique