Aberration-free calibration for 3D single molecule localization microscopy

We propose a straightforward sample-based technique to calibrate the axial detection in 3D single molecule localization microscopy (SMLM). Using microspheres coated with fluorescent molecules, the calibration curves of PSF-shaping- or intensity-based measurements can be obtained for any required depth range from a few hundreds of nanometers to several tens of microns. This experimental method takes into account the effect of the spherical aberration without requiring computational correction.Comment: 8 pages, 2 figures. Submitted to Optics Letters on October 12th, 201

Huntingtin proteolysis releases non-polyQ fragments that cause toxicity through dynamin 1 dysregulation

Cleavage of mutant huntingtin (HTT) is an essential process in Huntington's disease (HD), an inherited neurodegenerative disorder. Cleavage generates N-ter fragments that contain the polyQ stretch and whose nuclear toxicity is well established. However, the functional defects induced by cleavage of full-length HTT remain elusive. Moreover, the contribution of non-polyQ C-terminal fragments is unknown. Using time- and site-specific control of full-length HTT proteolysis, we show that specific cleavages are required to disrupt intramolecular interactions within HTT and to cause toxicity in cells and flies. Surprisingly, in addition to the canonical pathogenic N-ter fragments, the C-ter fragments generated, that do not contain the polyQ stretch, induced toxicity via dilation of the endoplasmic reticulum (ER) and increased ER stress. C-ter HTT bound to dynamin 1 and subsequently impaired its activity at ER membranes. Our findings support a role for HTT on dynamin 1 function and ER homoeostasis. Proteolysis-induced alteration of this function may be relevant to disease. Synopsis The development of a time and site-specifically controlled cleavage of the mutant huntingtin protein reveals a pathogenic mechanism induced by the non-polyQ-containing fragments that are generated upon proteolysis during disease progression. Huntingtin proteolysis generates N-ter fragments that contain the toxic polyQ stretch but also the corresponding C-ter fragments. N-ter to C-ter intramolecular interactions present in full-length huntingtin are abrogated by sequential cleavages. Whereas the N-ter polyQ fragments translocate into the nucleus, the non-polyQ C-ter huntingtin fragments remain in the cytoplasm and cause ER dilation, stress and cell death. C-ter huntingtin fragments bind and inactivate dynamin 1 at the ER thus causing ER dilation and toxicity. Site-specifically controlled cleavage of the mutant huntingtin protein reveals a pathogenic mechanism induced by non-polyQ-containing fragments that are generated upon proteolysis during disease progression.</p

Films et nanostructures métalliques pour l'exaltation de la fluorescence moléculaire

Rubrique : Thèses primées de la division de Chimie PhysiqueNational audienceMolecule fluorescence is subject to the influence of its direct electromagnetic environment. Excitation and emission processes can be modified by the presence of a metallic structure, due to interference phenomena and coupling to surface plasmon modes.Metallic films ranging in morphology from nanoparticles to percolated, continuous, plane and rough films were designed and characterized. Their influence on the optical behaviour of the fluorophores was investigated by experiments and a theoretical model, as a function of fluorophore-to-metal distance and molecular orientation. The detected signal is found to be amplified by one to two orders of magnitude. Moreover, fluorophore photostabilization and the modification of intermolecular energy transfer processes are reported.This paper demonstrates the interest in this technology for sensitivity improvement of DNA chip and for applications in cell and tissue imaging.La fluorescence d'une molécule est sensible à son environnement électromagnétique immédiat. La présence d'une structure métallique modifie l'excitation et l'émission des fluorophores, par le jeu d'interférences et la présence de modes de surface tels que les plasmons de surface. Dans cette perspective, des films métalliques de différentes morphologies ont été élaborés et caractérisés. Leur influence sur la réponse optique des fluorophores a été étudiée par la modélisation puis l'expérience, en fonction de paramètres comme la distance fluorophore/métal et l'orientation moléculaire. Une amplification importante du signal détecté est observée, ainsi qu'une photostabilisation des fluorophores et une modification des transferts d'énergie intermoléculaires. Cet article démontre l'intérêt de cette technologie pour améliorer la sensibilité dans les puces à ADN et pour l'imagerie des cellules et des tissus

Localisation nanométrique de molécules uniques par modulation du signal de fluorescence

La microscopie de localisation de molécules individuelles permet de dépasser la limite de diffraction, révélant ainsi l’organisation cellulaire à l’échelle nanométrique. Cette méthode reposant sur l’analyse spatiale du signal émis par les molécules, reste souvent limitée à l’observation d’objets biologiques à de faibles profondeurs, ou très peu aberrants. Nous montrons ici que l’introduction d’un paramètre temporel dans le processus de localisation via l’introduction d’une excitation modulée permet d’adresser ces limitations

Nanoroughened plasmonic films for enhanced biosensing detection

International audienceAlthough fluorescence is the prevailing labeling technique in biosensing applications, sensitivity improvement is still a striving challenge. We show that coating standard microscope slides with nanoroughened silver films provides a high fluorescence signal enhancement due to plasmonic interactions. As a proof of concept, we applied these films with tailored plasmonic properties to DNA microarrays. Using common optical scanning devices, we achieved signal amplifications of more than 40-fold

Dépasser la limite de diffraction en microscopie de fluorescence

La microscopie de fluorescence est un outil de référence dans l’étude des systèmes biologiques, alliant la spécificité offerte par la fluorescence et la possibilité d’un suivi non invasif en milieu vivant. Cependant comme l’ensemble des techniques de microscopie, elle est soumise au phénomène de diffraction introduit par l’objectif, qui limite la résolution de l’instrument. Ce texte présente les différentes méthodes permettant de dépasser cette contrainte en associant développement instrumental en optique et contrôle de la photophysique des fluorophores afin de révéler l’organisation cellulaire à l’échelle nanométrique