Résumé: Non-uniform illumination limits quantitative analyses of fluorescence imaging techniques. In particular, single molecule localization microscopy (SMLM) relies on high irradiances, but conventional Gaussian-shaped laser illumination restricts the usable field of view to around 40 microm x 40 microm. We present Adaptable Scanning for Tunable Excitation Regions (ASTER), a versatile illumination technique that generates uniform and adaptable illumination. ASTER is also highly compatible with optical sectioning techniques such as total internal reflection fluorescence (TIRF). For SMLM, ASTER delivers homogeneous blinking kinetics at reasonable laser power over fields-of-view up to 200 microm x 200 microm. We demonstrate that ASTER improves clustering analysis and nanoscopic size measurements by imaging nanorulers, microtubules and clathrin-coated pits in COS-7 cells, and beta2-spectrin in neurons. ASTER's sharp and quantitative illumination paves the way for high-throughput quantification of biological structures and processes in classical and super-resolution fluorescence microscopies.

Résumé: We demonstrate subwavelength axial sectioning on biological samples with a stimulated emission depletion (STED) microscope combined with supercritical angle fluorescence (SAF) detection. SAF imaging is a powerful technique for imaging the membrane of the cell based on the direct exploitation of the fluorophore emission properties. Indeed, only when fluorophores are close to the interface can their evanescent near-field emission become propagative and be detected beyond the critical angle. Therefore, filtering out the SAF emission from the undercritical angle fluorescence (UAF) emission in the back focal plane of a high-NA objective lens permits nanometer axial sectioning of fluorescent emitters close to the coverslip. When combined with STED microscopy, a straightforward gain in axial resolution can be reached without any alteration of the STED beam path. Indeed, STED-SAF implementation only requires a modification in the detection path of the STED microscope and thus could be widely implemented.

Résumé: Determining how cells generate and trans duce mechanical forces at the nanoscale is a major technical challenge for the understanding of numerous physiological and pathological processes. Podosomes are submicrometer cell structures with a columnar F-actin core surrounded by a ring of adhesion proteins, which possess the singular ability to protrude into and probe the extracellular matrix. Using protrusion force microscopy, we have previously shown that single podosomes produce local nanoscale protrusions on the extracellular environment. However, how cellular forces are distributed to allow this protruding mechanism is still unknown. To investigate the molecular machinery of protrusion force generation, we performed mechanical simulations and developed quantitative image analyses of nanoscale architectural and mechanical measurements. First, in silico modeling showed that the deformations of the substrate made by podosomes require protrusion forces to be balanced by local traction forces at the immediate core periphery where the adhesion ring is located. Second, we showed that three-ring proteins are required for actin polymerization and protrusion force generation. Third, using DONALD, a 3D nanoscopy technique that provides 20 nm isotropic localization precision, we related force generation to the molecular extension of talin within the podosome ring, which requires vinculin and paxillin, indicating that the ring sustains mechanical tension. Our work demonstrates that the ring is a site of tension, balancing protrusion at the core. This local coupling of opposing forces forms the basis of protrusion and reveals the podosome as a nanoscale autonomous force generator.

Résumé: 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.

Résumé: A new class of optoelectronic nanodevices consisting of 0D semiconductor nanocrystals and 2D single graphene layers is attracting much attention. In particular, such a system may be used to investigate and control the transfer of energy and charge in low-dimensional systems. To this end, the fluorescence dynamics of individual colloidal quantum dots(QDs) on graphene are investigated on both the 10−9 to 10−8 s time scale (fluorescence lifetime) and the 1−100 s time scale(blinking statistics) in this paper. We find that (i) a nonradiative energy transfer rate of ≈5 × 10+8 s−1 is obtained from the reduced lifetimes of QDs on graphene as opposed to those on insulating substrates such as glass; (ii) QDs still exhibit fluorescence intermittency (“blinking”) on graphene; (iii) the cumulative distribution functions of the “off” times may be described by power-law statistics; (iv) QD coupling to graphene increases the time spent in the “on” state while the time spent in the “off” state remains relatively unchanged; and (v) the fluorescence emission spectrum of the QDs is practically unaltered by the QD−graphene coupling.

Résumé: We present a new technique based on the self-interference of Supercritical Angle Fluorescence (SAF) emission in order to perform full-field cell membrane imaging. We show that our Point Spread Function (PSF) engineering technique allows us to obtain a 100 nm axial sectioning while conserving the original lateral resolution of the microscope. The images are acquired using an optical module that can be connected to any fluorescent microscope to simultaneously monitor in real time both the cell membrane and in-depth phenomena.

Résumé: In this paper, we discuss the possibility of making a super-axially-resolved image of a biological sample using supercritical angle diffusion. This labeling-free approach is suitable to any microscope equipped with a N A(obj) > 1.33 microscope objective and can be used either for conventional intensity imaging or for quantitative phase imaging. We expose some results on beads an cells showing the potential of this method.

Résumé: Biocompatible, near-infrared luminescent gold nanoclusters (AuNCs) are synthesized directly in water using poly(ethylene glycol)-dithiolane ligands terminating in either a carboxyl, amine, azide, or methoxy group. The ≈1.5 nm diameter AuNCs fluoresce at ≈820 nm with quantum yields that range from 4–8%, depending on the terminal functional group present, and display average luminescence lifetimes approaching 1.5 μs. The two-photon absorption (TPA) cross-section and two-photon excited fluorescence (TPEF) properties are also measured. Long-term testing shows the poly(ethylene glycol) stabilized AuNCs maintain colloidal stability in a variety of media ranging from saline to tissue culture growth medium along with tolerating storage of up to 2 years. DNA and dye-conjugation reactions confirm that the carboxyl, amine, and azide groups can be utilized on the AuNCs for carbodiimide, succinimidyl ester, and CuI-assisted cycloaddition chemistry, respectively. High signal-to-noise one- and two-photon cellular imaging is demonstrated. The AuNCs exhibit outstanding photophysical stability during continuous-extended imaging. Concomitant cellular viability testing shows that the AuNCs also elicit minimal cytotoxicity. Further biological applications for these luminescent nanoclustered materials are discussed.

Résumé: We report a new full-field fluorescence microscopy method for imaging live cell membranes based on supercritical near-field emission. This technique consists of extracting the self-interference between undercritical and supercritical light by simple image subtraction. In the objective back focal plane, this is equivalent to adding a virtual mask blocking the subcritical emission. We show that this virtual mask is radically different from a real physical mask, enabling a 100 nm axial confinement and enhancing the image sensitivity without damaging the lateral resolution. This technique is easy to implement and simultaneously provides images of the inner parts of the cell and its membrane with standard-illumination light.

Résumé: Full-field optical coherence microscopy (FF-OCM) and optically sectioned fluorescence microscopy are two imaging techniques that are implemented here in a novel dual modality instrument. The two imaging modalities use a broad field illumination to acquire the entire field of view without raster scanning. Optical sectioning is achieved in both imaging modalities owing to the coherence gating property of light for FF-OCM, and a structured illumination setup for fluorescence microscopy. Complementary image data are provided by the dual modality instrument in the context of biological tissue screening. FF-OCM imaging modality shows the tissue microarchitecture, while fluorescence microscopy highlights specific tissue features with cellular-level resolution by using targeting contrast agents. Complementary tissue morphology and biochemical features could potentially improve the understanding of cellular functions and disease diagnosis. (C) 2012 Optical Society of America

Résumé: In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. They play a role in cellular physiological processes such as signalling, and trafficking but are also thought to be key players in several diseases including viral or bacterial infections and neurodegenerative diseases. Yet their existence is still a matter of controversy. Indeed, lipid raft size has been estimated to be around 20 nm, far under the resolution limit of conventional microscopy (around 200 nm), thus precluding their direct imaging. Up to now, the main techniques used to assess the partition of proteins of interest inside lipid rafts were Detergent Resistant Membranes (DRMs) isolation and co-patching with antibodies. Though widely used because of their rather easy implementation, these techniques were prone to artefacts and thus criticized. Technical improvements were therefore necessary to overcome these artefacts and to be able to probe lipid rafts partition in living cells. Here we present a method for the sensitive analysis of lipid rafts partition of fluorescently-tagged proteins or lipids in the plasma membrane of living cells. This method, termed Fluorescence Correlation Spectroscopy (FCS), relies on the disparity in diffusion times of fluorescent probes located inside or outside of lipid rafts. In fact, as evidenced in both artificial membranes and cell cultures, probes would diffuse much faster outside than inside dense lipid rafts. To determine diffusion times, minute fluorescence fluctuations are measured as a function of time in a focal volume (approximately 1 femtoliter), located at the plasma membrane of cells with a confocal microscope (Fig. 1). The auto-correlation curves can then be drawn from these fluctuations and fitted with appropriate mathematical diffusion models. FCS can be used to determine the lipid raft partitioning of various probes, as long as they are fluorescently tagged. Fluorescent tagging can be achieved by expression of fluorescent fusion proteins or by binding of fluorescent ligands. Moreover, FCS can be used not only in artificial membranes and cell lines but also in primary cultures, as described recently. It can also be used to follow the dynamics of lipid raft partitioning after drug addition or membrane lipid composition change.

Résumé: Amyloid peptide (A beta) is generated by sequential cleavage of the amyloid precursor protein (APP) by beta-secretase (Bace1) and gamma-secretase. A beta production increases after plasma membrane cholesterol loading through unknown mechanisms. To determine how APP-Bace1 proximity affects this phenomenon, we developed a fluorescence lifetime imaging microscopy-Forster resonance energy transfer (FLIM-FRET) technique for visualization of these molecules either by epifluorescence or at the plasma membrane only using total internal reflection fluorescence. Further, we used fluorescence correlation spectroscopy to determine the lipid rafts partition of APP-yellow fluorescent protein (YFP) and Bace1-green fluorescent protein (GFP) molecules at the plasma membrane of neurons. We show that less than 10 min after cholesterol exposure, Bace1-GFP/APP-mCherry proximity increases selectively at the membrane and APP relocalizes to raft domains, preceded by rapid endocytosis. After longer cholesterol exposures, APP and Bace1 are found in proximity intracellularly. We demonstrate that cholesterol loading does not increase A beta production by having a direct impact on Bace1 catalytic activity but rather by altering the accessibility of Bace1 to its substrate, APP. This change in accessibility is mediated by clustering in lipid rafts, followed by rapid endocytosis.-Marquer, C., Devauges, V., Cossec, J.-C., Liot, G., Lecart, S., Saudou, F., Duyckaerts, C., Leveque-Fort, S., Potier, M.-C. Local cholesterol increase triggers amyloid precursor protein-Bace1 clustering in lipid rafts and rapid endocytosis. FASEB J. 25, 1295-1305 (2011). www.fasebj.org

Résumé: We present the instrumental development of a versatile total internal reflection fluorescence lifetime imaging microscopy setup illuminated by a supercontinuum laser source. It enables performing wide-field fluorescence lifetime. imaging with subwavelength axial resolution for a large range of fluorophores. The short overall acquisition time and the axial resolution are well suited for dynamic neurobiological applications. (C) 2009 Optical Society of America

Résumé: Metallic films and nanostructures for molecular fluorescence enhancement Molecule 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.

Résumé: We present the generation of a picosecond polarized supercontinuum in highly birefringent multimodal microstructured fiber. The initial steps of the spectral broadening are dominated by intermodal four-wave mixing controlled by the specific fiber design. Using a low repetition rate ultra-stable solid state laser, a pulse train well-suited for versatile time-domain fluorescence lifetime imaging applications is obtained. (C) 2008 Optical Society of America

Résumé: Two-photon fluorescence microscopy is widely applied to biology and medicine to study both the structure and dynamic processes in living cells. The main issue is the slow acquisition rate due to the point scanning approach limiting the multimodal detection (x, y, z, t). To extend the performances of this powerful technique, we present a time-resolved multifocal multiphoton microscope (MMM) based on laser amplitude splitting. An array of 8 X 8 foci is created on the sample that gives a direct insight of the fluorescence localization. Four-dimensional (4D) imaging is obtained by combining simultaneous foci scanning, time-gated detection, and z displacement. We illustrate time-resolved MMM capabilities for 4D imaging of a photosensitizer inside living colon cancer cells. The aim of this study is to have a better understanding of the photophysical processes implied in the photosensitizer reactivity. (C) 2007 Optical Society of America.

Résumé: Fluorescence microscopy has become the method of choice in the majority of life-science applications. We describe development and use of mirror slides to significantly enhance the fluorescence signal using standard air microscope objectives. This technique offers sufficient gain to achieve high-sensitivity imaging, together with wide field of observation and large depth of focus, two major breakthroughs for routine analysis and high-throughput screening applications on cells and tissue samples. (C) 2007 Society of Photo-Optical Instrumentation Engineers.

Résumé: Received July 22, 2005; revised September 12, 2005; accepted September 14, 2005 We demonstrate, for the first time, to our knowledge, a diode-pumped passively mode-locked Nd:YVO4 laser, operating on the F-4(3/2) I-_4(9/2) transition of the neodymium ion at 914 nm. We obtained 8.8 ps pulses at approximately 914 nm at a repetition rate of 94 MHz, and an averaged output power of 87 mW by using a semiconductor saturable absorber mirror. (c) 2006 Optical Society of America.

Résumé: Fluorescence correlation spectroscopy (FCS) under two-photon excitation was used successfully to characterize the diffusion properties of model virus particles (bacteriophages) in bacterial biofilm of Stenotrophonas maltophilia. The results are compared to those obtained with fluorescent latex beads used as a reference. The FCS data clearly demonstrated the possibility for viral particles to penetrate inside the exopolymeric matrix of mucoid biofilms, and hence to benefit from its protective effect toward antimicrobials (antibiotics and biocides). Microbial biofilms should hence be considered as potential reservoirs of pathogenic viruses, and are probably responsible for numerous persistent viral infections.