Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy.

Essential cellular functions as diverse as genome maintenance and tissue morphogenesis rely on the dynamic organization of filamentous assemblies. For example, the precise structural organization of DNA filaments has profound consequences on all DNA-mediated processes including gene expression, whereas control over the precise spatial arrangement of cytoskeletal protein filaments is key for mechanical force generation driving animal tissue morphogenesis. Polarized fluorescence is currently used to extract structural organization of fluorescently labeled biological filaments by determining the orientation of fluorescent labels, however with a strong drawback: polarized fluorescence imaging is indeed spatially limited by optical diffraction, and is thus unable to discriminate between the intrinsic orientational mobility of the fluorophore labels and the real structural disorder of the labeled biomolecules.

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

The 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.

Septins promote F-actin ring formation by crosslinking actin filaments into curved bundles.

Animal cell cytokinesis requires a contractile ring of crosslinked actin filaments and myosin motors. How contractile rings form and are stabilized in dividing cells remains unclear. We address this problem by focusing on septins, highly conserved proteins in eukaryotes whose precise contribution to cytokinesis remains elusive.

Mapping the local organization of cell membranes using excitation-polarization-resolved confocal fluorescence microscopy.

Fluorescence anisotropy and linear dichroism imaging have been widely used for imaging biomolecular orientational distributions in protein aggregates, fibrillar structures of cells, and cell membranes. However, these techniques do not give access to complete orientational order information in a whole image, because their use is limited to parts of the sample where the average orientation of molecules is known a priori. Fluorescence anisotropy is also highly sensitive to depolarization mechanisms such as those induced by fluorescence energy transfer.

High frame-rate fluorescence confocal angle-resolved linear dichroism microscopy.

Angle-resolved linear dichroism is a recent technique that exploits images recorded using an illumination field whose polarization angle is sequentially rotated during acquisition. It allows to retrieve orientation information of the fluorescent molecules, namely the average orientation angle and the amplitude of the fluctuations around this average. In order to boost up the acquisition speed without sacrificing the axial sectioning, we propose to combine a spinning disk confocal excitation scheme together with an electrooptical polarization switching and a camera acquisition.

Probing orientational behavior of MHC class I protein and lipid probes in cell membranes by fluorescence polarization-resolved imaging.

Steady-state polarization-resolved fluorescence imaging is used to analyze the molecular orientational order behavior of rigidly labeled major histocompatibility complex class I (MHC I) proteins and lipid probes in cell membranes of living cells. These fluorescent probes report the orientational properties of proteins and their surrounding lipid environment. We present a statistical study of the molecular orientational order, modeled as the width of the angular distribution of the molecules, for the proteins in the cell endomembrane and plasma membrane, as well as for the lipid probes in the plasma membrane.

A versatile dual spot laser scanning confocal microscopy system for advanced fluorescence correlation spectroscopy analysis in living cell.

A fluorescence correlation spectroscopy system based on two independent measurement volumes is presented. The optical setup and data acquisition hardware are detailed, as well as a complete protocol to control the location, size, and shape of the measurement volumes. A method that allows to monitor independently the excitation and collection efficiency distribution is proposed.