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Supplement 1

International audience; Among the multitude of optical polarization contrasts that can be observed in complex biological specimens, linear diattenuation (LD) imaging has received little attention. It is indeed challenging to image LD with basic polarizing microscopes because it is often relatively small in comparison with linear retardance (LR). In addition, interpretation of LD images is not straightforward when experiments are conducted in the visible range because LD can be produced by both dichroism and anisotropic scattering. Mueller polarimetry is a powerful implementation of polarization sensing able to differentiate and measure the anisotropies of specimens. In this article, near infrared transmission Mueller scanning microscopy is used to image LD in thin biological specimen sections made of various proteins with unprecedented resolution and sensitivity. The near infrared spectral range makes it possible to lower the contribution of dichroism to the total linear diattenuation in order to highlight anisotropic scattering. Pixel-by-pixel comparison of LD images with LR and multiphoton images demonstrates that LD is produced by under-resolved structures that are not revealed by other means, notably within the sarcomere of skeletal muscles. LD microscopy appears as a powerful tool to provide new insights into the macro-molecular organization of biological specimens at the sub-microscopic scale without labelling.

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Mueller microscopes enable imaging of the optical anisotropic properties of biological or non-biological samples, in phase and amplitude, at sub-micrometer scale. However, the development of Mueller microscopes faces instrumental challenges: whilst adjusting the microscope, the operator needs a polarimetric image as guidance and the production of polarimetric parameters must be sufficiently quick to ensure fast imaging. To mitigate this issue, in this paper, a full Mueller scanning microscope based on spectral encoding of polarization is presented. The spectrum collected every 10 ms for each position of the optical beam on the specimen, incorporates all the information needed to produce the full Mueller matrix, which allows simultaneous images of all the polarimetric parameters at the unequalled rate of 1.5 Hz (for an image of 256×256 pixels). The design of the optical blocks allows for the real-time display of linear birefringent images which serve as guidance for the operator. In addition, the instrument has the capability to easily switch its functionality from a Mueller to a Second Harmonic Generation (SHG) microscope, providing a pixel-to-pixel matching of the images produced by the two modalities. The device performance is illustrated by imaging various unstained biological specimens.

International audience; Polarization-resolved second harmonic generation (P-SHG) microscopy is able to probe the sub-micrometer structural organization of myosin filaments within skeletal muscle. In this study, P-SHG microscopy was used to analyze the structural consequences of sepsis, which is the main cause of the critical illness polyneuromyopathy (CIPNM). Experiments conducted on two populations of rats demonstrated a significant difference of the anisotropy parameter between healthy and septic groups, indicating that P-SHG microscopy is promising for the diagnosis of CIPNM. The difference, which can be attributed to a change of myosin conformation at the sub-sarcomere scale, cannot be evidenced by classical SHG imaging.

Mueller matrix polarimeters allow the acquisition of the full linear polarimetric response of a medium, and lead to the knowledge of its dichroism, birefringence and depolarization properties. By now, those setups used to generate the multiple states of polarization necessary for the calculation of a Mueller matrix in a sequential way, which limits their acquisition time at the millisecond scale. In this work, we propose the development of the first experimental Mueller matrix polarimeter with a spectral encoding of the polarization, so as to parallelize the generation of polarization's states. The acquisition time of a Mueller matrix is thus limited by the acquisition time of the detector, and could be very short. The aim of this thesis was, first of all, the experimental validation of the first Mueller matrix polarimeter by wavelength polarization coding, which was named the "snapshot Mueller matrix polarimeter". The instrument use a broadband source (10nm), high order retarders and a spectrometer coupled with a CCD camera. The calibration of the instrument was done, and relies upon a detailed modeling of the optical elements at play. The precision of the polarimeter was estimated to 3%, thanks to measurements on well known media. The stabilization of the polarimeter was also considered, in order to take into account the deviations due to temperature changes, and strains applied on the optical fiber. Finally, two theoretical ways of optimization were proposed: the ideal choice of the thickness configuration for the retarders and the use of a two-channel polarimeter. The instrument was then used to perform static and dynamic characterization of ferroelectric liquid crystal cells (pure and polymer-stabilized), in the SSFLC geometry. It first proved the benefits of the exploitation of Mueller matrices for the evaluation of structures in such samples, thanks to numerous parameters. Then, those studies proved the feasibility of following fast dynamic reorientations ( ∼ 100μs ) with a simple and rapid tool. Particularly, we showed that the smectic layers reorient during the "up"/"down" transition of a SSFLC cell. Keys words: optic, instrumentation characterization, polarisation, polarimetry, Mueller matrix, calibration,; ilieu, dans le but de déterminer ses propriétés de dichroïsme, de biréfringence et de dépolarisation. Jusqu'à présent, ces instruments utilisaient une génération séquentielle des multiples états de polarisation nécessaires au calcul d'une matrice de Mueller, leur temps d'acquisition étant donc limité à la milliseconde. Nous proposons dans cette thèse de développer le premier polarimètre de Mueller dont le codage de la polarisation est effectué dans le domaine spectral, ce qui permet une génération parallèle des états de polarisation. Le temps d'acquisition d'une matrice de Mueller est alors limité par le temps d'intégration du détecteur, qui peut être potentiellement très court. L'objet de la thèse a été dans un premier temps de valider expérimentalement un polarimètre de Mueller par codage en longueur d'onde, que nous avons dénommé "le polarimètre de Mueller instantané". Il s'agit d'utiliser une source large bande (10nm), des lames de phases d'ordre élevé et un spectromètre couplé à une caméra CCD pour disperser tous les états de polarisation. L'étalonnage de l'instrument a été effectué, et s'appuie sur une modélisation détaillée des composants optiques utilisés dans le montage. La précision du polarimètre a été évaluée à 3%, grâce à des mesures sur des milieux connus. La stabilisation du polarimètre a également été envisagée, pour prendre en compte les dérives dues à la température et aux contraintes appliquées à la fibre optique. Enfin, deux pistes théoriques d'optimisation ont été proposées, à savoir le choix idéal de la configuration d'épaisseur des lames de phase et l'utilisation d'un polarimètre à deux voies de détection. L'instrument a ensuite été utilisé pour caractériser de manière statique et dynamique des cellules à cristaux liquides ferroélectriques (pure et stabilisée par polymère) en dispositif SSFLC. Cela a permis d'une part, de prouver que l'exploitation de la matrice de Mueller est avantageuse pour évaluer des organisations moléculaires dans ces échantillons, grâce à la variété d'information qu'elle propose. Puis, ces études ont également prouvé la faisabilité d'acquérir simplement et avec une bonne résolution temporelle des dynamiques de réorientations rapides ( ∼ 100μs ) avec le polarimètre instantané. Nous avons ainsi été capables en particulier de mettre en évidence le mouvement des couches smectiques lors d'une transition "up"/"down" dans une cellule SSFLC.