Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

International audienceFast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces photo-bleaching and phototoxic effects compared to confocal microscopy, while providing 3D images with subcellular resolution. Here we present the setup of a light sheet based microscope, which is composed of an upright microscope and a small set of opto-mechanical elements for the generation of the light sheet. The protocol describes how to build, align the microscope and characterize the light sheet. In addition, it details how to implement the method for in toto imaging of C. elegans embryos using a simple observation chamber. The method allows the capture of 3D two-colors time-lapse movies over few hours of development. This should ease the tracking of cell shape, cell divisions and tagged proteins over long periods of time

Molecular clustering in the cell: from weak interactions to optimized functional architectures

International audienceMolecular components of the cell, such as lipids, proteins or RNA molecules, can associate through weak interactions and form clusters. A growing number of studies have shown that clustering of molecules is crucial for cell functions such as signal optimization and polarization. Clustering provides an intermediate level of organization between the molecular and cellular scales. Here we review recent studies focusing on how molecular clustering functions in different biological contexts, the potential importance of clustering for information processing, as well as the physical nature of cluster formation. We mainly refer to literature focusing on clusters within cell membranes, but also report findings on clusters in the cytosol, emphasizing their ubiquitous role

Laser Ablation to Probe the Epithelial Mechanics in Drosophila

International audienceLaser ablation is nowadays a widespread technique to probe tissue mechanics during development. Here we describe the setup of one such ablation system and ablation experiments performed on the embryo and pupa of Drosophila. We describe in detail the process of sample preparation, how to disrupt single-cell junctions and perform linear or circular cuts at the tissue scale, and how to analyze the data to determine relevant mechanical parameters

Physics puzzles on membrane domains posed by cell biology.

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Superresolution measurements in vivo: imaging Drosophila embryo by photoactivated localization microscopy

International audienceVisualization and quantification of supramolecular assemblies in cells are essential to understand the design principles of cells and tissues. The advent of photoactivated localization microscopy (PALM) and related techniques has offered unprecedented information on protein supramolecular assemblies in 3-D with a spatial resolution of a few tens of nanometers. Yet application of PALM microscopy for in vivo studies remains challenging. This chapter describes how to implement PALM microscopy for quantitative analysis of intercellular adhesion in the Drosophila embryo. Our protocol describes the sample preparation, the imaging setup, and the acquisition procedure. We also discuss how to proceed with quantitative analysis of data. Initially designed and implemented for Drosophila embryo imaging of intercellular adhesion, this protocol can be readily adapted to other structures than adhesions and other organisms such as Zebrafish or Caenorhabditis elegans

Membrane microdomains: from seeing to understanding

International audienceThe plasma membrane is a composite material, which forms a semi-permeable barrier and an interface for communication between the intracellular and extracellular environments. While the existence of membrane microdomains with nanoscale organization has been proved by the application of numerous biochemical and physical methods, direct observation of these heterogeneities using optical microscopy has remained challenging for decades, partly due to the optical diffraction limit, which restricts the resolution to ~200 nm. During the past years, new optical methods which circumvent this fundamental limit have emerged. Not only do these techniques allow direct visualization, but also quantitative characterization of nanoscopic structures. We discuss how these emerging optical methods have refined our knowledge of membrane microdomains and how they may shed light on the basic principles of the mesoscopic membrane organization

Measuring forces and stresses in situ in living tissues

International audienceDevelopment, homeostasis and regeneration of tissues result from a complex combination of genetics and mechanics, and progresses in the former have been quicker than in the latter. Measurements of in situ forces and stresses appear to be increasingly important to delineate the role of mechanics in development. We review here several emerging techniques: contact manipulation, manipulation using light, visual sensors, and non-mechanical observation techniques. We compare their fields of applications, their advantages and limitations, and their validations. These techniques complement measurements of deformations and of mechanical properties. We argue that such approaches could have a significant impact on our understanding of the development of living tissues in the near future.© 2016. Published by The Company of Biologists Ltd

Planar polarized actomyosin contractile flows control epithelial junction remodelling

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Détection et exaltation de la luminescence de molécules biologiques individuelles en solution

La Spectroscopie de Corrélation de Fluorescence (FCS) est une technique d'analyse statistique des fluctuations de luminescence produites par des molécules fluorescentes diffusant librement dans un volume de collection de quelque $\mu$m$^3$. Une limitation fondamentale de la technique provient de l'ouverture numérique limitée des systèmes optiques conventionnels qui ne collectent qu'une faible partie des photons émis. Nous présentons des résultats relatifs à l'augmentation du nombre de photons collectés en utilisant le concept du contrôle de l'émission spontanée par des structures photoniques de type miroir diélectrique