Imaging of Red-Shifted Light From Bioluminescent Tumors Using Fluorescence by Unbound Excitation From Luminescence

Early detection of tumors is today a major challenge and requires sensitive imaging methodologies coupled with new efficient probes. In vivo optical bioluminescence imaging has been widely used in the field of preclinical oncology to visualize tumors and several cancer cell lines have been genetically modified to provide bioluminescence signals. However, the light emitted by the majority of commonly used luciferases is usually in the blue part of the visible spectrum, where tissue absorption is still very high, making deep tissue imaging non-optimal, and calling for optimized optical imaging methodologies. We have previously shown that red-shifting of bioluminescence signal by Fluorescence Unbound Excitation from Luminescence (FUEL) is a mean to increase bioluminescence signal sensitivity detection in vivo. Here, we applied FUEL to tumor detection in two different subcutaneous tumor models: the auto-luminescent human embryonic kidney (HEK293) cell line and the murine B16-F10 melanoma cell line previously transfected with a plasmid encoding the Luc2 firefly luciferase. Tumor size and bioluminescence were measured over time and tumor vascularization characterized. We then locally injected near infrared emitting Quantum Dots (NIR QDs) in the tumor site and observed a red-shifting of bioluminescence signal by (FUEL) indicating that FUEL could be used to allow deeper tumor detection in mice

Détection résolue en temps de quantum dots infrarouge pour le suivi de cellules

In vivo cell tracking is a promising tool to improve our understanding of certain biological processes (circulating tumour cells migration, immune cells activity). Fluorescence microscopy ensures a high resolution and a good sensitivity. The latter is however limited by the high tissue autofluorescence and poor visible light penetration depth.We present the synthesis and characterization of Zn-Cu-In-Se / ZnS (core/shell) QDs made of low toxicity materials. These QDs exhibit a bright emission centered around 800 nm, where absorption and scattering of tissues are minimal. These nanocrystals are coated with a new surface chemistry, which yields small and bright probes in the cell cytoplasm for several days after labelling.These QDs also present a fluorescence lifetime much longer (150 ns) than the tissue autofluorescence (<5 ns). By combining a pulsed excitation source to a time-gated fluorescence imaging system, we show that we can efficiently discriminate the intracellular QDs signal from autofluorescence in an ex vivo sample and thus increase the detection sensitivity of labelled cells into tissues. We also report preliminary results obtained in an in vivo sample.Le suivi de cellules in vivo est essentiel afin de déterminer par exemple les voies de migration de cellules tumorales circulantes ou encore pour suivre l’activité de cellules immunitaires. La microscopie de fluorescence assure une bonne résolution ainsi qu’une grande sensibilité et semble adaptée à la détection de cellules uniques in vivo dans un modèle de souris. Néanmoins, sa sensibilité est limitée par deux principaux facteurs: le signal d’autofluorescence des tissus d’une part, et l’absorption et la diffusion de la lumière visible dans les tissus d’autre part.Nous présentons la synthèse et la caractérisation optique des quantum dots Zn-Cu-In- Se/ZnS composés de matériaux peu toxiques. Ces nanoparticules possèdent un maximum d’émission centré autour de 800 nm, où l’absorption et la diffusion par les tissus biologiques sont limitées. Elles sont rendues biocompatibles grâce à une chimie de surface développée au laboratoire qui permet d’obtenir des sondes petites, brillantes et stables en milieu intracellulaire pendant plusieurs jours.Grâce à leur temps de vie fluorescence beaucoup plus long (150 ns) que celui de l’ autofluorescence des tissus (< 5 ns), nous avons développé un microscope de fluorescence résolu en temps qui permet de sélectionner le signal d’une cellule isolée marquée par des quantum dots et rejeter l’autofluorescence du tissu environnant. Nous présentons également les premières images in vivo de veines marquées avec ces sondes et observées avec notre microscope

In Vivo Imaging of Single Tumor Cells in Fast-Flowing Bloodstream Using Near-Infrared Quantum Dots and Time-Gated Imaging

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Multimodal Mn-doped I-III-VI quantum dots for near infrared fluorescence and magnetic resonance imaging: from synthesis to in vivo application.

International audienceThe development of sensitive multimodal contrast agents is a key issue to provide better global, multi-scale images for diagnostic or therapeutic purposes. Here we present the synthesis of Zn-Cu-In-(S, Se)/Zn(1-x)Mn(x)S core-shell quantum dots (QDs) that can be used as markers for both near-infrared fluorescence imaging and magnetic resonance imaging (MRI). We first present the synthesis of Zn-Cu-In-(S, Se) cores coated with a thick ZnS shell doped with various proportions of Mn. Their emission wavelengths can be tuned over the NIR optical window suitable for deep tissue imaging. The incorporation of manganese ions (up to a few thousand ions per QD) confers them a paramagnetic character, as demonstrated by structural analysis and electron paramagnetic resonance spectroscopy. These QDs maintain their optical properties after transfer to water using ligand exchange. They exhibit T1-relaxivities up to 1400 mM(-1) [QD] s(-1) at 7 T and 300 K. We finally show that these QDs are suitable multimodal in vivo probes and demonstrate MRI and NIR fluorescence detection of regional lymph nodes in mice

Sulfobetaine–Vinylimidazole Block Copolymers: A Robust Quantum Dot Surface Chemistry Expanding Bioimaging’s Horizons

Long-term inspection of biological phenomena requires probes of elevated intra- and extracellular stability and target biospecificity. The high fluorescence and photostability of quantum dot (QD) nanoparticles contributed to foster their promise as bioimaging tools that could overcome limitations associated with traditional fluorophores. However, QDs’ potential as a bioimaging platform relies upon a precise control over the surface chemistry modifications of these nano-objects. Here, a zwitterion–vinylimidazole block copolymer ligand was synthesized, which regroups all anchoring groups in one compact terminal block, while the rest of the chain is endowed with antifouling and bioconjugation moieties. By further application of an oriented bioconjugation approach with whole IgG antibodies, QD nanobioconjugates were obtained that display outstanding intra- and extracellular stability as well as biorecognition capacity. Imaging the internalization and intracellular dynamics of a transmembrane cell receptor, the CB1 brain cannabinoid receptor, both in HEK293 cells and in neurons, illustrates the breadth of potential applications of these nanoprobes

Ultrarare heterozygous pathogenic variants of genes causing dominant forms of early-onset deafness underlie severe presbycusis

International audiencePresbycusis, or age-related hearing loss (ARHL), is a major public health issue. About half the phenotypic variance has been attributed to genetic factors. Here, we assessed the contribution to presbycusis of ultrarare pathogenic variants, considered indicative of Mendelian forms. We focused on severe presbycusis without environmental or comorbidity risk factors and studied multiplex family age-related hearing loss (mARHL) and simplex/sporadic age-related hearing loss (sARHL) cases and controls with normal hearing by whole-exome sequencing. Ultrarare variants (allele frequency [AF] < 0.0001) of 35 genes responsible for autosomal dominant early-onset forms of deafness, predicted to be pathogenic, were detected in 25.7% of mARHL and 22.7% of sARHL cases vs. 7.5% of controls ( P = 0.001); half were previously unknown (AF < 0.000002). MYO6 , MYO7A , PTPRQ , and TECTA variants were present in 8.9% of ARHL cases but less than 1% of controls. Evidence for a causal role of variants in presbycusis was provided by pathogenicity prediction programs, documented haploinsufficiency, three-dimensional structure/function analyses, cell biology experiments, and reported early effects. We also established Tmc1 N321I/+ mice, carrying the TMC1 :p.(Asn327Ile) variant detected in an mARHL case, as a mouse model for a monogenic form of presbycusis. Deafness gene variants can thus result in a continuum of auditory phenotypes. Our findings demonstrate that the genetics of presbycusis is shaped by not only well-studied polygenic risk factors of small effect size revealed by common variants but also, ultrarare variants likely resulting in monogenic forms, thereby paving the way for treatment with emerging inner ear gene therapy