Quantitative analysis of light scattering in polarization-resolved nonlinear microscopy

International audiencePolarization resolved nonlinear microscopy (PRNM) is a powerful technique to gain microscopic structural information in biological media. However, deep imaging in a variety of biological specimens is hindered by light scattering phenomena, which not only degrades the image quality but also affects the polarization state purity. In order to quantify this phenomenon and give a framework for polarization resolved microscopy in thick scattering tissues, we develop a characterization methodology based on four wave mixing (FWM) process. More specifically, we take advantage of two unique features of FWM, meaning its ability to produce an intrinsic in-depth local coherent source and its capacity to quantify the presence of light depolarization in isotropic regions inside a sample. By exploring diverse experimental layouts in phantoms with different scattering properties, we study systematically the influence of scattering on the nonlinear excitation and emission processes. The results show that depolarization mechanisms for the nonlinearly generated photons are highly dependent on the scattering center size, the geometry used (epi/forward) and, most importantly, on the thickness of the sample. We show that the use of an un-analyzed detection makes the polarization-dependence read-out highly robust to scattering effects, even in regimes where imaging might be degraded. The effects are illustrated in polarization resolved imaging of myelin lipid organization in mouse spinal cord

Catheter-directed therapy to treat intermediate- and high-risk pulmonary embolism: Personal experience and review of the literature

Pulmonary embolism (PE) is the third leading cause of cardiovascular death in the western world. Prompt recognition, risk stratification, and individualized treatment are crucial to improve outcomes in patients with PE. Anticoagulation alone is a sufficient therapeutic option in low-risk patients, whereas primary reperfusion with systemic thrombolysis (ST) is usually chosen in high-risk patients. The choice of treatment in intermediate-risk patients is complex and depends on the clinical presentation. Catheter-directed therapy (CDTh) includes all therapies delivered via a catheter placed in the branches of the pulmonary arteries directly into the thrombus. Because ST bears a high risk of major bleeding and numerous patients have contraindications to ST, CDTh is an alternative to ST in intermediate- and high-risk PE patients. CDTh includes local thrombolysis using low-dose alteplase, ultrasound-assisted thrombolysis, and mechanical fragmentation and aspiration of the thrombi, as well as their combinations. In this review article, we have summarized devices and technical details for CDTh, discussed the efficacy and safety of CDTh in comparison to ST in previous clinical trials, and outlined future research directions regarding CDTh, both based on the literature and our personal experience from the local PE Response Team of the Center for the Management of Pulmonary Embolism (CELZAT) in Warsaw

Microscopie multimodale non-linéaire résolue en polarisation pour l’étude des lipides: modèles membranes à la myéline dans les tissus.

Polarization resolved nonlinear microscopy is a powerful tool to image structural information in biomolecular assemblies. Nonlinear interaction between light and matter lead to complex processes where coherent combinations of optical fields couple to assemblies of molecular transition dipoles. Controlling polarized optical fields and monitoring nonlinear induced signals in a medium can nevertheless bring rich information on molecular orientational organization within the focal spot of a microscope ob jective. In this PhD thesis we apply this polarization sensitivity to different label-free optical coherent techniques (coherent anti-Stokes Raman scattering (CARS), stimulated Raman scattering (SRS)) and to two-photon fluorescence (2PEF) to retrieve quantitative information on the staticmolecular distribution shape and orientation of lipids in model membranes and biological membranes such as myelin sheaths in spinal cord tissues. With this technique, we address fundamental questions about lipid packing behavior in membranes, and how it can be affected by other molecules such as cholesterol and the insertion of fluorescent lipidprobes. We demonstrate that polarization resolved CARS give access to fine details on lipids arrangement in myelin sheaths, at a sub-diffraction scale. In the context of experimental autoimmune encephalomyelitis disease (EAE) we show, that even at the stage of disruption of the myelin envelope during the demyelination process, lipids multilayersreveal strong capability to preserve their macroscopic self-assembly into highly organized structures, with a degree of disorganization occurring only at the molecular scale. At last, we show that such technique can be combined with a larger scope of studies in bioimaging; we use multimodal microscopy (combined CARS and 2PEF) to image axonaldegeneration and immune cell recruitment associated to demyelination process in order to address biological questions related to the immune response and axon damage during progression of the neurodegenerative disease.La microscopie non-linéaire résolue en polarisation est un outil puissant pour accéder à des informations structurelles dans les assemblages biomoléculaires. Les interactions non-linéaire entre matière et lumière induisent des processus complexes où des champs électromagnétiques cohérents interagissent avec les dipôles de transitions moléculaires. Le contrôle de la polarisation des champs électromagnétiques excitateurs et l’étude desréponses non-linéaires induites procurent de riches informations sur la distribution angulaire des molécules présentes dans le volume focal de l’objectif du microscope. Dans cette thèse, nous appliquons cette sensibilité à la polarisation à plusieurs modalités de microscopie cohérentes sans marquage (diffusion cohérente Raman anti-Stokes (CARS), diffusion Cohérente stimulée (SRS)) et à la fluorescence à deux photons (2PEF) afind’obtenir des informations quantitatives sur la forme de la distribution moléculaire et l’orientation des lipides dans les membranes artificielles, ainsi que dans les membranes biologiques telles que la myéline des tissues de la moelle épinière. Avec cette technique, nous adressons une question fondamentale sur le comportement des ensembles lipidiques dans les membranes et sur l’effet d’autres molécules telles que le cholestérol et les marqueursfluorescents. Nous démontrons que le CARS résolu en polarisation permet d’accéder à de fines informations sur l’organisation des lipides dans les membranes de la myéline, en deçà de la limite de diffraction. Dans le contexte de l’encéphalomyélite allergique expérimentale (EAE), nous démontrons que même au stade de la perturbation de la couche de myéline lors du processus de démyélinisation, les multicouches de lipides présentent une fortecapacité de préserver leur auto-assemblage macroscopique en présentant une structure hautement organisée avec un niveau de désorganisation qui ne se retrouve qu’à l’échelle moléculaire. Finalement, nous démontrons qu’une telle technique peut être combinée à d’autres pour un plus grand éventail d’études en bio-imagerie; nous utilisons la microscopie multimodale (combinaison de CARS et de 2PEF) pour imager la dégénération des axones et le recrutement de cellules immunitaires associé au processus de démyélinisation afin d’adresser des questions biologiques reliées à la réponse immunitaire et les dommages des axones durant la progression des maladies neurodégénératives

Lipids−Fluorophores Interactions Probed by Combined Nonlinear Polarized Microscopy

International audienc

Lipids–Fluorophores Interactions Probed by Combined Nonlinear Polarized Microscopy

International audienc

In vivo single human sweat gland activity monitoring using coherent anti-Stokes Raman scattering and two-photon excited auto-fluorescence microscopy

International audienc