Structure of molecular packing probed by polarization-resolved nonlinear four-wave mixing and coherent anti-Stokes Raman-scattering microscopy

International audienceWe report a method that is able to provide refined structural information on molecular packing in biomolecular assemblies using polarization-resolved four-wavemixing and coherent anti-Stokes Raman-scattering microscopy. These third-order nonlinear processes allow quantifying high orders of symmetry which are exploited here to reveal a high level of detail in the angular disorder behavior at the molecular scale in lipid membranes

Ultimate use of two-photon fluorescence microscopy to map orientational behavior of fluorophores

International audienceThe orientational distribution of fluorophores is an important reporter of the structure and function of their molecular environment. Although this distribution affects the fluorescence signal under polarized-light excitation, its retrieval is limited to a small number of parameters. Because of this limitation, the need for a geometrical model (cone, Gaussian, etc.) to effect such retrieval is often invoked. In this work, using a symmetry decomposition of the distribution function of the fluorescent molecules, we show that polarized two-photon fluorescence based on tunable linear dichroism allows for the retrieval of this distribution with reasonable fidelity and without invoking either an a priori knowledge of the system to be investigated or a geometrical model. We establish the optimal level of detail to which any distribution can be retrieved using this technique. As applied to artificial lipid vesicles and cell membranes, the ability of this method to identify and quantify specific structural properties that complement the more traditional molecular-order information is demonstrated. In particular, we analyze situations that give access to the sharpness of the angular constraint, and to the evidence of an isotropic population of fluorophores within the focal volume encompassing the membrane. Moreover, this technique has the potential to address complex situations such as the distribution of a tethered membrane protein label in an ordered environment

Thioflavine-T and Congo Red reveal the polymorphism of insulin amyloid fibrils when probed by polarization-resolved fluorescence microscopy.

International audienceAmyloid fibrils are protein misfolding structures that involve a β-sheet structure and are associated with the pathologies of various neurodegenerative diseases. Here we show that Thioflavine-T and Congo Red, two major dyes used to image fibrils by fluorescence assays, can provide deep structural information when probed by means of polarization-resolved fluorescence microscopy. Unlike fluorescence anisotropy or fluorescence detected linear dichroism imaging, this technique allows to retrieve simultaneously both mean orientation and orientation dispersion of the dye, used here as a reporter of the fibril structure. We have observed that insulin amyloid fibrils exhibit a homogeneous behavior over the fibrils' length, confirming their structural uniformity. In addition, these results reveal the existence of various structures among the observed fibrils' population, in spite of a similar aspect when imaged with conventional fluorescence microscopy. This optical nondestructive technique opens perspectives for in vivo structural analyses or high throughput screening

Developments in the Photonic Theory of Fluorescence

Conventional fluorescence commonly arises when excited molecules relax to their ground electronic state, and most of the surplus energy dissipates in the form of photon emission. The consolidation and full development of theory based on this concept has paved the way for the discovery of several mechanistic variants that can come into play with the involvement of laser input – most notably the phenomenon of multiphoton-induced fluorescence. However, other effects can become apparent when off-resonant laser input is applied during the lifetime of the initial excited state. Examples include a recently identified scheme for laser-controlled fluorescence. Other systems of interest are those in which fluorescence is emitted from a set of two or more coupled nanoemitters. This chapter develops a quantum theoretical outlook to identify and describe these processes, leading to a discussion of potential applications ranging from all-optical switching to the generation of optical vortices

Intensity Weighted Subtraction Microscopy Approach for Image Contrast and Resolution Enhancement

We propose and demonstrate a novel subtraction microscopy algorithm, exploiting fluorescence emission difference or switching laser mode and their derivatives for image enhancement. The key novelty of the proposed approach lies in the weighted subtraction coefficient, adjusted pixel-by-pixel with respect to the intensity distributions of initial images. This method produces significant resolution enhancement and minimizes image distortions. Our theoretical and experimental studies demonstrate that this approach can be applied to any optical microscopy techniques, including label free and non-linear methods, where common super-resolution techniques cannot be used

Randomized controlled trial protocol to investigate the antiplatelet therapy effect on extracellular vesicles (AFFECT EV) in acute myocardial infarction

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Probing molecular order in zeolite L inclusion compounds using two-photon fluorescence polarimetric microscopy

We investigate the local static molecular orientational behavior in zeolite L inclusion compounds by polarimetric two-photon fluorescence microscopy. This technique, based on the polarized read-out of the signal under a tunable incident polarization state, provides refined information on molecular disorder that is not achievable using traditional fluorescence anisotropy. Moreover, the polarimetric microscopy imaging scheme permits a spatial investigation of possible heterogeneities, with a submicrometric resolution. The study performed on different fluorescent molecules inserted in zeolite L channels evidence a degree of disorder for either small or flexible structures