Julien Douady

Crystal growth and refined Sellmeier equations over the complete transparency range of RbTiOPO4

Abstract The phase-matching directions sum- and difference-frequency generations are measured in the principal planes of RbTiOPO 4 crystals grown from a halide flux. The use of crystals with a cylindrical shape and of a tunable laser source allows us to perform accurate measurements over the complete transparency range of that material, and to determine a refined set of Sellmeier equations valid for any phase-matched interaction in that crystal.

Shape-Based Tracking Allows Functional Discrimination of Two Immune Cell Subsets Expressing the Same Fluorescent Tag in Mouse Lung Explant

Dendritic Cells (DC) represent a key lung immune cell population, which play a critical role in the antigen presenting process and initiation of the adaptive immune response. The study of DCs has largely benefited from the joint development of fluorescence microscopy and knock-in technology, leading to several mouse strains with constitutively labeled DC subsets. However, in the lung most transgenic mice do express fluorescent protein not only in DCs, but also in closely related cell lineages such as monocytes and macrophages. As an example, in the lungs of CX3CR1+/gfp mice the green fluorescent protein is expressed mostly by both CD11b conventional DCs and resident monocytes. Despite this non-specific staining, we show that a shape criterion can discriminate these two particular subsets. Implemented in a cell tracking code, this quantified criterion allows us to analyze the specific behavior of DCs under inflammatory conditions mediated by lipopolysaccharide on lung explants. Compared to monocytes, we show that DCs move slower and are more confined, while both populations do not have any chemotactism-associated movement. We could generalize from these results that DCs can be automatically discriminated from other round-shaped cells expressing the same fluorescent protein in various lung inflammation models.

Calculation of quadratic cascading contributions associated with a phase-matched cubic frequency difference generation in a KTiOPO4 crystal

This study deals with the calculation of the contributions of quadratic cascading processes which are coupled with a cubic frequency conversion interaction phase-matched in a non-centrosymmetric crystal. We have considered the case of a difference frequency generation interaction in KTiOPO4 in order to determine the phase-matching directions with the lowest quadratic cascading rate, the target being to ensure a cubic process that is as pure as possible. These calculations can be applied to any other crystal.

Symmetry and phase-matching properties of third-harmonic generation under the photoelastic effect in Ge-As-Se chalcogenide glasses

We discuss the study of three chalcogenide glasses of high third-order nonlinear electric susceptibility, AsSe4,GeSe4, and GeAsSe8. We measured the strain optical coefficients, and we determined dispersion equations of the refractive index from previously published measurements. From these data, we performed a theoretical study of a new scheme for third-harmonic generation in a glass where phase matching is created by a photoelastic effect. A complete symmetry analysis based on Curie principle allowed us to define the configuration of polarization of the interacting waves, leading to a nonzero cubic effective coefficient. The generated third-harmonic intensity is then calculated.

An optimized two-photon method for in vivo lung imaging reveals intimate cell collaborations during infection

Lung tissue motion arising from breathing and heart beating has been described as the largest annoyance of in vivo imaging. Consequently, infected lung tissue has never been imaged in vivo thus far, and little is known concerning the kinetics of the mucosal immune system at the cellular level. We have developed an optimized post-processing strategy to overcome tissue motion, based upon two-photon and second harmonic generation (SHG) microscopy. In contrast to previously published data, we have freed the lung parenchyma from any strain and depression in order to maintain the lungs under optimal physiological parameters. Excitation beams swept the sample throughout normal breathing and heart movements, allowing the collection of many images. Given that tissue motion is unpredictably, it was essential to sort images of interest. This step was enhanced by using SHG signal from collagen as a reference for sampling and realignment phases. A normalized cross-correlation criterion was used between a manually chosen reference image and rigid transformations of all others. Using CX3CR1+/gfp mice this process allowed the collection of high resolution images of pulmonary dendritic cells (DCs) interacting with Bacillus anthracis spores, a Gram-positive bacteria responsible for anthrax disease. We imaged lung tissue for up to one hour, without interrupting normal lung physiology. Interestingly, our data revealed unexpected interactions between DCs and macrophages, two specialized phagocytes. These contacts may participate in a better coordinate immune response. Our results not only demonstrate the phagocytizing task of lung DCs but also infer a cooperative role of alveolar macrophages and DCs.

Multimodal 4D imaging of cell-pathogen interactions in the lungs provides new insights into pulmonary infections

Lung efficiency as gas exchanger organ is based on the delicate balance of its associated mucosal immune system between inflammation and sterility. In this study, we developed a dynamic imaging protocol using confocal and twophoton excitation fluorescence (2PEF) on freshly harvested infected lungs. This modus operandi allowed the collection of important information about CX3CR1+ pulmonary cells. This major immune cell subset turned out to be distributed in an anisotropic way in the lungs: subpleural, parenchymal and bronchial CX3CR1+ cells have then been described. The way parenchymal CX3CR1+ cells react against LPS activation has been considered using Matlab software, demonstrating a dramatic increase of average cell speed. Then, interactions between Bacillus anthracis spores and CX3CR1+ dendritic cells have been investigated, providing not only evidences of CX3CR1+ cells involvement in pathogen uptake but also details about the capture mechanisms.

Multiphoton microscopy as a diagnostic tool for pathological analysis of sentinel lymph nodes

Multiphoton microscopy has shown a powerful potential for biomedical in vivo and ex vivo analysis of tissue sections and explants. Studies were carried out on several animal organs such as brain, arteries, lungs, and kidneys. One of the current challenges is to transfer to the clinic the knowledge and the methods previously developed in the labs at the preclinical level. For tumour staging, physicians often remove the lymph nodes that are localized at the proximity of the lesion. In case of breast cancer or melanoma, sentinel lymph node protocol is performed: pathologists randomly realize an extensive sampling of formol fixed nodes. However, the duration of this protocol is important and its reliability is not always satisfactory. The aim of our study was to determine if multiphoton microscopy would enable the fast imaging of lymph nodes on important depths, with or without exogenous staining. Experiments were first conducted on pig lymph nodes in order to test various dyes and to determine an appropriate protocol. The same experiments were then performed on thin slices of human lymph nodes bearing metastatic melanoma cells. We obtained relevant images with both endofluorescence plus second-harmonic generation and xanthene dyes. They show a good contrast between tumour and healthy cells. Furthermore, images of pig lymph nodes were recorded up to 120μm below the surface. This new method could then enable a faster diagnosis with higher efficiency for the patient. Experiments on thicker human lymph nodes are currently underway in order to validate these preliminary results.

Imaging elastic and collagen fibers with sulforhodamine B and second-harmonic generation

Since the early nineties, multiphoton microscopy has become a powerful tool to investigate morphological and physiological parameters in vivo or on thick ex vivo sections. To stain structures of interest many dyes have been developed and two-photon properties (cross section, excitation and emission spectra) of existing ones have been characterized. Recently, our team has shown a new property of sulforhodamine B (SRB). This dye has the ability to bind specifically elastic fibers. The observation of elastin using its endofluorescence properties was already widely described but required long exposition delays up to 10s and the imaging depth was limited to approximately 50 μm. With a multiphoton microscope and SRB, it is possible to observe elastic fibers directly in the living animal or on thick tissue sections with a micrometric spatial resolution in less than one second per image with an imaging depth of ~ 200 μm. Moreover, with an appropriate set of filters, we can acquire simultaneously the SRB and the second harmonic generation (SHG) signals of collagen fibers. Here, we report various applications of this new staining method on different arterial rings. The layers of the arterial wall, as well as, the elastic lamellae are observed and are numbered. With the addition of a nuclear stain such as the Hoechst 33342, a more accurate morphological study of the arterial walls can be accomplished. Finally, an intravital observation of the saphenous artery morphology is presented.