Guillaume Dupuis

Pigment Identification by Fiber-Optics Diffuse Reflectance Spectroscopy

Fiber-optics reflectance spectroscopy is used to identify pigments in pictorial layers of works of art thanks to a spectra database of dry powdered mineral pigments. Measurements are noninvasive, without any contact, and can be implemented in situ, without moving the work of art under investigation from its conservation place. The experimental device, using the special back-scattering configuration, is briefly presented. The protocol leading to the constitution of the spectra database of dry mineral pigments is described. Unlike other studies, this protocol has been developed to emphasize multiple scattering of light by elementary pigments in comparison with specular reflection on the surface of the sample. In these conditions, the diffuse reflectance spectrum is the label of the mineral pigment. The numerical processing of pigment identification is detailed. Both the influences of the roughness of the studied surface and of a possible varnish layer are taken into account when numerical identification is implemented. Several applications on patrimonial works of art are reported.

Direct optical nanoscopy with axially localized detection

Evanescent light excitation is widely used in super-resolution fluorescence microscopy to confine light and reduce background noise. Here, we propose a method of exploiting evanescent light in the context of emission. When a fluorophore is located in close proximity to a medium with a higher refractive index, its near-field component is converted into light that propagates beyond the critical angle. This so-called supercritical-angle fluorescence can be captured using a high-numerical-aperture objective and used to determine the axial position of the fluorophore with nanometre precision. We introduce a new technique for three-dimensional nanoscopy that combines direct stochastic optical reconstruction microscopy (dSTORM) with dedicated detection of supercritical-angle fluorescence emission. We demonstrate that our approach of direct optical nanoscopy with axially localized detection (DONALD) typically yields an isotropic three-dimensional localization precision of 20 nm within an axial range of ∼150 nm above the coverslip. Researchers exploit direct stochastic optical reconstruction microscopy and dedicated detection of super-critical-angle fluorescence emission to enable direct optical nanoscopy with axially localized detection.

Simultaneous optically sectioned fluorescence and optical coherence microscopy with full-field illumination

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.

Towards an optical biopsy for the diagnosis of breast cancer in vivo by endogenous fluorescence spectroscopy

The techniques of medical imaging allow the detection of suspect lesions in the breast, but they do not always evidence the malignant nature of these lesions. Breast biopsies and histological analyses are therefore implemented to establish a diagnosis. In order to reduce the number of these invasive procedures, a portable clinical system was designed based upon the excitation of Endogenous Fluorescence in vivo at 405 nm via a fiber-optics probe included in a disposable needle of small diameter (<1 mm). From the fluorescence signal, the authors are able to discriminate between diseased and healthy areas of human breast biopsies. Stronger fluorescence intensity and systematic spectral red shift of the tumor areas were observed. These results are confirmed by confocal microscopy. This new instrument is promising for the minimally invasive diagnosis of breast tumors in vivo with an appreciable limitation of patient trauma and of operational and financial cost.

Confocal supercritical angle microscopy for cell membrane imaging

We demonstrate subwavelength sectioning on biological samples with a conventional confocal microscope. This optical sectioning is achieved by the phenomenon of supercritical angle fluorescence, wherein only a fluorophore next to the interface of a refractive index discontinuity can emit propagating components of radiation into the so-called forbidden angles. The simplicity of this technique allows it to be integrated with a high numerical aperture confocal scanning microscope by only a simple modification on the detection channel. Confocal-supercritical angular fluorescence microscopy would be a powerful tool to achieve high-resolution surface imaging, especially for membrane imaging in biological samples.

Podosome Force Generation Machinery: A Local Balance between Protrusion at the Core and Traction at the Ring

Determining how cells generate and transduce 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.

Combining 3D single molecule localization strategies for reproducible bioimaging

We developed a 3D localization-based super-resolution technique providing an almost isotropic 3D resolution over a 1 µm range with precisions down to 15 nm. The axial localization is performed through a combination of point spread function (PSF) shaping and supercritical angle fluorescence (SAF), which yields absolute axial information. Using a dual-view scheme, the axial detection is decoupled from the lateral detection and optimized independently. This method can be readily implemented on most homemade PSF shaping setups and provides drift-free, tilt-insensitive and achromatic results. Its insensitivity to these unavoidable experimental biases is especially adapted for multicolor 3D super-resolution microscopy, as we demonstrate by imaging cell cytoskeleton, living bacteria membranes and axon periodic submembrane scaffolds. We further illustrate the interest of the technique for biological multicolor imaging over a several µm range by direct merging of multiple acquisitions at different depths.

BIN1 genetic risk factor for Alzheimer is sufficient to induce early structural tract alterations in entorhinal cortex-dentate gyrus pathway and related hippocampal multi-scale impairments

Late-onset Alzheimer Disease (LOAD) is the most common form of dementia and one of the most challenging diseases of modern society 1. Understanding the preclinical stages of AD that begins in the brain at least 2-3 decades before evidence of episodic memory defects in patients is pivotal for the design of successful approaches to delay or reverse the transition from normal brain function to cognitive impairments. Our working hypothesis is that LOAD genetic risk factors can be sufficient to generate early phenotypical changes before any changes in either Abeta or Tau. To test this hypothesis, we generated an hBIN1 mouse model based on the human BIN1 gene overexpression that we found in post-mortem brain samples from LOAD patients. BIN1 is the second important risk factor for AD, following the APOE gene 2. We identified co-deregulated gene repertoires common to both 7-week mouse hippocampus sub-regions and post-mortem brain samples from LOAD patients, demonstrating the validity of this hBIN1 model. We evidenced an early phenotype of neurodegeneration starting at 3 months with structural impairment fiber pathways quantified by high resolution (17.2T) (MRI-DTI) and related functional impacts. We found structural changes in entorhinal cortex-dentate gyrus (EC-DG) pathway known to be the earliest brain region impacted in LOAD 3–6. Similarly, the function of this pathway was impaired both in vitro and in vivo, with the changes in spine density and dendritic simplification of DG neurons, impaired EC-DG long-term potentiation (LTP) and behavioral deficits linked to object recognition episodic memory. As expected for a neurodegenerative model, we evidenced the progression of dysfunction at the morphological, functional and behavioral levels with age. Structural spreading involved impairment of fibers in somatosensory and temporal associative cortexes at month 15. Functional and behavioral spreading was characterized by impact on pattern separation of spatial episodic memory. Moreover, this neurodegeneration occurred without any detectable changes in Aβ42 and tau. Overall, these data show the possibility to identify a repertoire of molecular changes occurring both in patients and in hBIN1 mice and whose further manipulation can be instrumental to rescue or delay episodic memory defects.

Failure of daptomycin to kill Staphylococcus aureus: impact of bacterial membrane fatty acid composition

ABSTRACT Daptomycin is a last-resort membrane-targeting lipopeptide approved for the treatment of drug-resistant staphylococcal infections, such as bacteremia and implant-related infections. Although cases of resistance to this antibiotic are rare, increasing numbers of clinical, in vitro, and animal studies report treatment failure, notably against Staphylococcus aureus. The aim of this study was to identify the features of daptomycin and its target bacteria that lead to daptomycin treatment failure. We show that daptomycin bactericidal activity against S. aureus varies significantly with the growth state and strain, according to the membrane fatty acid composition. Daptomycin efficacy as an antibiotic relies on its ability to oligomerize within membranes and form pores that subsequently lead to cell death. Our findings ascertain that daptomycin interacts with tolerant bacteria and reaches its membrane target, regardless of its bactericidal activity. However, the final step of pore formation does not occur in cells that are daptomycin tolerant, strongly suggesting that it is incapable of oligomerization. Importantly, membrane fatty acid contents correlated with poor daptomycin bactericidal activity, which could be manipulated by fatty acid addition. In conclusion, daptomycin failure to treat S. aureus is not due to a lack of antibiotic-target interaction, but is driven by its capacity to form pores, which depends on membrane composition. Manipulation of membrane fluidity to restore S. aureus daptomycin bactericidal activity in vivo could open the way to novel antibiotic treatment strategies.

Time-resolved wide-field optically sectioned fluorescence microscopy

We present the implementation of a fast wide-field optical sectioning technique called HiLo microscopy on a fluorescence lifetime imaging microscope. HiLo microscopy is based on the fusion of two images, one with structured illumination and another with uniform illumination. Optically sectioned images are then digitally generated thanks to a fusion algorithm. HiLo images are comparable in quality with confocal images but they can be acquired faster over larger fields of view. We obtain 4D imaging by combining HiLo optical sectioning, time-gated detection, and z-displacement. We characterize the performances of this set-up in terms of 3D spatial resolution and time-resolved capabilities in both fixed- and live-cell imaging modes.