Application of Synchrotron Radiation-Based Micro-Analysis on Cadmium Yellows in Pablo Picasso's Femme

The cultural heritage community is increasingly exploring synchrotron radiation (SR) based techniques for the study of art and archaeological objects. When considering heterogeneous and complex micro-samples, such as those from paintings, the combination of different SR X-ray techniques is often exploited to overcome the intrinsic limitations and sensitivity of the single technique. Less frequently, SR X-ray analyses are combined with SR micro-photoluminescence or micro-Fourier Transform Infrared spectroscopy, which provide complementary information on the molecular composition, offering a unique integrated analysis approach. Although the spatial correlation between the maps obtained with different techniques is not straightforward due to the different volumes probed by each method, the combination of the information provides a greater understanding and insight into the paint chemistry. In this work, we discuss the advantages and disadvantages of the combination of X-ray techniques and SR-based photoluminescence through the study of two paint micro-samples taken from Pablo Picasso's Femme (1907). The painting contains two cadmium yellow paints (based on CdS): one relatively intact and one visibly degraded. SR micro-analyses demonstrated that the two Cd-yellow paints differ in terms of structure, chemical composition, and photoluminescence properties. In particular, on the basis of the combination of different SR measurements, we hypothesize that the degraded yellow is based on nanocrystalline CdS with high presence of Cd(OH)Cl. These two characteristics have enhanced the reactivity of the paint and strongly influenced its stability

A framework for dissecting affinities of multidrug efflux transporter AcrB to fluoroquinolones

Sufficient concentration of antibiotics close to their target is key for antimicrobial action. Among the tools exploited by bacteria to reduce the internal concentration of antibiotics, multidrug efflux pumps stand out for their ability to capture and expel many unrelated compounds out of the cell. Determining the specificities and efflux efficiency of these pumps towards their substrates would provide quantitative insights into the development of antibacterial strategies. In this light, we developed a competition efflux assay on whole cells, that allows measuring the efficacy of extrusion of clinically used quinolones in populations and individual bacteria. Experiments reveal the efficient competitive action of some quinolones that restore an active concentration of other fluoroquinolones. Computational methods show how quinolones interact with the multidrug efflux transporter AcrB. Combining experiments and computations unveils a key molecular mechanism acting in vivo to detoxify bacterial cells. The developed assay can be generalized to the study of other efflux pumps

Bioimaging of liver cancer cells incubated with partially reduced graphene oxide

Functional materials based on graphene oxide (GO) and reduced graphene oxide (rGO) have a high potential for application in the fields of biophysics, material science, and biomedical engineering [1]. It is due to their tunable physical properties, high surface area, remarkable photoluminescence, as well as their controllable chemical functionalization [2]. Beyond their applications in nanomedicine for drug/gene delivery, phototherapy and bioimaging, they have shown significant interaction and adhesive properties with proteins, mammalian cells and microorganisms, which makes them potential candidates for multifunctional biological applications. In this lecture, we will present a study of the interaction of partially reduced graphene oxide (prGO) with Huh7.5.1 liver cancer cells. The study was conducted by means of synchrotron excitation DUV fluorescence bioimaging (performed on DISCO beamline of synchrotron SOLEIL) [3]. The prGO sample was obtained by the reduction (to a certain extent) of the initially prepared GO nanosheets. The fluorescence of the GO nanosheets increases with time of the reduction due to a change in the ratio of the sp2 and sp3 carbon sites, and the prGO sample was extracted from the dispersion when the intensity of the fluorescence reached its maximum. After that, Huh7.5.1 cells were incubated with GO, prGO and rGO nanosheets and used in bioimaging studies. The presence of graphene materials influenced the fluorescence properties of the cells, and by analyzing fluorescence photobleaching dynamics, we were able to localize graphene nanosheets inside the liver cancer cells.VII International School and Conference on Photonics : PHOTONICA2019 : Abstracts of Tutorial, Keynote, Invited Lectures, Progress Reports and Contributed Papers; August 26-30; Belgrad

A fluorescent nanoprobe for single bacterium tracking: functionalization of silver nanoparticles with tryptophan to probe the nanoparticle accumulation with single cell resolution

The investigation of the interaction of silver nanoparticles and live bacteria cells is of particular importance for understanding and controlling their bactericidal properties. In this study, the process of internalization of silver nanoparticles in Escherichia coli cells was followed by means of synchrotron excitation deep ultraviolet (DUV) fluorescence imaging. Antimicrobial nanostructures that can absorb and emit light in the UV region were prepared by functionalization of silver nanoparticles with tryptophan amino acid and used as environmentally sensitive fluorescent probes. The nanostructures were characterized by morphological (TEM) and spectroscopic methods (UV-vis, FTIR, XPS, and photoluminescence). The TEM images and the analyses of the UV-vis spectra suggested that the addition of tryptophan led to the formation of hybrid nanostructures with pronounced eccentricity and larger sizes with respect to that of the initial silver nanoparticles. The DUV imaging showed that it was possible to distinguish the fluorescent signal pertaining to silver-tryptophan nanostructures from the autofluorescence of the bacteria. The spatial resolution of the fluorescence images was 154 nm which was sufficient to perform analyses of the accumulation of the nanostructures within a single bacterium. The DUV imaging results imply that the tryptophan-functionalized silver nanoparticles interact with cell membranes via insertion of the amino acid into the phospholipid bilayer and enter the cells

A New Synchrotron Approach to Study Ancient Materials: UV/Visible Photoluminescence Micro-Imaging

International audienc

Towards the in situ study of bacterial single cells following freeze-drying using synchrotron radiation

Communication orale sur invitationTowards the in situ study of bacterial single cells following freeze-drying using synchrotron radiation. 6. International Conference of Lyophilization and Freeze-Dryin

Photosensitizer effects on cancerous cells: A combined study using synchrotron infrared and fluorescence microscopies

International audienceHypocrellin A (HA), a lipid-soluble peryloquinone derivative, isolated from natural fungus sacs of Hypocrella bambusae, has been reported to be a highly potential photosensitizer in photodynamic therapy (PDT). It has been studied increasingly because of its anticancer activities when irradiated with light. We have studied the interaction mechanisms of HA with HeLa cells as a function of incubation time. Fluorescence microscopy confirmed that HA localisation is limited in the cytoplasm before eventually concentrating in clusters around the nucleus. The IR spectra of HA-treated, PDT-treated and control HeLa cells were recorded at the ESRF Infrared beamline (ID21). Principal component analysis has been used to assess the IR spectral changes between the various HeLa cells spectral data sets (The Unscrambler software, CAMO). PCA revealed that there is a frequency shift of protein amide I and amide II vibrational bands, indicating changes in the protein secondary structures of the HA-treated and PDT-treated cancer cells compared to the control cells. In addition, the relative DNA intensity in HA-treated cells decreases gradually along the incubation time. The use of synchrotron infrared microscopy is shown to be of paramount importance for targeting specifically the biochemical modification induced in the cell nucleus

Deep UV excited muscle cell autofluorescence varies with the fibre type

International audienceThe rat skeletal muscle consists of four pure types of muscle cells called type I, type IIA, type IIX and type IIB, and their hybrids in different proportions. They differ in their contraction speeds and metabolic pathways. The intracellular composition is adapted to the fibre function and therefore to fibre types. Given that small differences in composition are likely to alter the optical properties of the cells, we studied the impact of the cell type on the fluorescence response following excitation in the deep UV region. Rat soleus and extensor digitorum longus (EDL) muscle fibres, previously identified based on their cell types by immunohistofluorescence analysis, were analyzed by synchrotron fluorescence microspectroscopy on stain-free serial muscle cross-sections. Muscle fibres excited at 275 nm showed differences in the fluorescence emission intensity among fibre types at 302, 325, 346 and 410 nm. The 410/325 ratio decreased significantly with contractile and metabolic features in EDL muscle, in the order of I \\textgreater IIA \\textgreater IIX \\textgreater IIB fibres (p \\textless 0.01). Compared to type I fibres, the 346/302 ratio of IIA fibres decreased significantly in both EDL and soleus muscles (p \\textless 0.01). This study highlights the usefulness of autofluorescence spectral signals to characterize histological cross-sections of muscle fibres with no staining chemicals

Pixel categorization based on resonance energy transfer between fluorescent molecules: a pathway towards localization of functionalized metal nanoparticles in individual cells by fluorescence microscopy

Molecular resonance energy transfer (RET) is a basis of a half-century-old tool known as the molecular ruler, used for inference of mutual distances of two spectrally-coupled fluorophores usually relevant in biological contexts [1]. RET influences not only the absolute intensities of fluorescence signals of the molecular pair, but also their other optical characteristics, e.g. the valence electrons’ excited states. On the other hand, metal-based hybrid nanostructures show great promise as future multifunctional nanoplatforms, due to their superior sensitivity to external perturbations [2]. The metal components of the nanostructures influence the optical properties of fluorescent molecules in the vicinity, similarly as bulk metals do, which often presents a drawback. Here we want to demonstrate how this particular feature in metal-based nanosystems can be used as an advantage point in conventional biophysical imaging if the beneficial underlying physical mechanisms are identified [3]. In this talk, we will first explain the influence of gold nanoparticles on resonance energy transfer between tryptophan, essential amino acid, and riboflavin, vitamin B2, in an aqueous environment. We will show that the relative change of the signal in the tryptophan spectral channel is more pronounced when the biomolecules are attached to the nanoparticles. Afterward, we will demonstrate how timelapse fluorescence imaging of nanoparticle-incubated liver cancer cells can be used for inferring subcellular areas of nanoparticle accumulation. By exposing the incubated cell to continuous UV radiation, the photobleaching of the fluorescent molecules in the sample occurs. Being sensitive to the electronic structure of the fluorophore, the photobleaching rates of different subareas in the cell show distinctive trends, depending on the chemical composition. As the nanoparticles promote RET between tryptophan and riboflavin, the nanostructures exhibit a particular photobleaching trend compared to the cells' endogenous fluorescent species [4]. Thus, by separating areas of fluorescence images into different classes based on their photobleaching rates, i.e. grouping the pixels of the same signal trends, we are able to infer the preferential accumulation sites of bifunctionalized gold nanoparticles by using fluorescence microscopy of lower lateral resolution than the size of the nanoparticles. In this way, we demonstrate a potent auxiliary detection modality of metal-based hybrid nanostructures in optical imaging.VIII International School and Conference on Photonics and HEMMAGINERO workshop : PHOTONICA2021 : book of abstracts; August 23-27, 2021; Belgrad