25 research outputs found
AxonSeg: open source software for axon and myelin segmentation and morphometric analysis
Segmenting axon and myelin from microscopic images is relevant for studying the peripheral and central nervous system and for validating new MRI techniques that aim at quantifying tissue microstructure. While several software packages have been proposed, their interface is sometimes limited and/or they are designed to work with a specific modality (e.g., scanning electron microscopy (SEM) only). Here we introduce AxonSeg, which allows to perform automatic axon and myelin segmentation on histology images, and to extract relevant morphometric information, such as axon diameter distribution, axon density and the myelin g-ratio. AxonSeg includes a simple and intuitive MATLABbased graphical user interface (GUI) and can easily be adapted to a variety of imaging modalities. The main steps of AxonSeg consist of: (i) image pre-processing; (ii) pre-segmentation of axons over a cropped image and discriminant analysis (DA) to select the best parameters based on axon shape and intensity information; (iii) automatic axon and myelin segmentation over the full image; and (iv) atlas-based statistics to extract morphometric information. Segmentation results from standard optical microscopy (OM), SEM and coherent anti-Stokes Raman scattering (CARS) microscopy are presented, along with validation against manual segmentations. Being fully-automatic after a quick manual intervention on a cropped image, we believe AxonSeg will be useful to researchers interested in large throughput histology
Polarimetric multiphoton fluorescence microscopy in molecular and biological media
Light-matter interaction in molecular and bio-molecular media can lead to complex processes where optical fields polarizations couple to an assembly of molecular transition dipoles. The manipulation of the optical fields polarization in fluorescence microscopy can in particular give access to fine changes occurring in molecular arrangements. In this PhD thesis we report a method based on a tuneable excitation polarization state complemented by a polarized read-out, applied to polarization-resolved multiphoton fluorescence microscopy. Two-photon fluorescence polarimetry allows to retrieve a quantitative information on the static molecular distribution shape and orientation in different environments such as model lipid membranes, cell membranes, and molecular inclusion compounds that can be strongly heterogeneous. Three-photon fluorescence polarimetry has been furthermore applied in bio-molecular media in order to provide a diagnostics for crystallinity in protein crystals with high sensitivity to their structure and symmetry. The experimental implementation of polarimetric multi-photon microscopy requires to quantify possible polarization distortions originating from the experimental set-up or sample itself, which are thoroughly analyzed.Les interactions lumière-matière dans les mileux moléculaires et bio-moléculaires peuvent mener à des processus complexes où les polarisations des champs optiques se couplent aux assemblages de dipoles de transitions moléculaires. La manipulation des polarisations des champs optiques en microscopie de uorescence peut en particulier donner accès à des modi cations nes d'arrangements moléculaires. Dans ce travail de thèse nous introduisons une méthode basée sur la variation continue d'un état de polarisation d'excitation complémentée par une analyse polarisée, appliquée à la microscopie de uorescence multi-photons. La uorescence à deux photons polarimétrique permet d'accéder à une information statique quantitative sur la forme et l'orientation de la distribution orientationnelle moléculaire dans des membranes lipidiques articielles, dans des cellules ou sur des composés molécluaires co-cristallins qui peuvent être fortement hétérogènes. La uorescence à trois photons polarimétrique apporte de plus un diagnostique de cristallinité dans des cristaux de protéines, avec une forte sensibilité à leur structure et symétrie. L'implémentation expérimentale de cette technique requiert de quantier les distortions de polarisation provenant du montage expérimental et de l'échantillon lui-même, qui sont nement analysés
Microscopie non-linéaire polarimétrique dans les milieux moléculaires et biologiques
Les interactions lumière-matière dans les mileux moléculaires et bio-moléculaires peuvent mener à des processus complexes où les polarisations des champs optiques se couplent aux assemblages de dipoles de transitions moléculaires. La manipulation des polarisations des champs optiques en microscopie de fluorescence peut en particulier donner accès à des modifications fines d'arrangements moléculaires. Dans ce travail de thèse nous introduisons une méthode basée sur la variation continue d'un état de polarisation d'excitation complémentée par une analyse polarisée, appliquée à la microscopie de fluorescence multi-photons. La fluorescence à deux photons polarimétrique permet d'accéder à une information statique quantitative sur la forme et l'orientation de la distribution orientationnelle moléculaire dans des membranes lipidiques artificielles, dans des cellules ou sur des composés molécluaires co-cristallins qui peuvent être fortement hétérogènes. La fluorescence à trois photons polarimétrique apporte de plus un diagnostique de cristallinité dans des cristaux de protéines, avec une forte sensibilité à leur structure et symétrie. L'implémentation expérimentale de cette technique requiert de quantifier les distortions de polarisation provenant du montage expérimental et de l'échantillon lui-même, qui sont finement analysés.AIX-MARSEILLE3-BU Sc.St Jérô (130552102) / SudocSudocFranceF
Polarimetric two-fluorescence microscopy for investigation of molecular order in lipid environment
Investigation of molecular and protein crystals by three photon polarization resolved microscopy.
International audienceWe implement three photon fluorescence polarization resolved microscopy to optically investigate molecular and protein crystals. The availability of UV transitions using IR pulses allows analyses without fluorescence staining. Polarization resolved studies indicate that high-order symmetry structures can be revealed and that strong fluorescent energy transfer occurs between molecules. We show how this permits identification of a monocrystalline nature for a sample at the subwavelength resolution scale
AxonSeg: open source software for axon and myelin segmentation and morphometric analysis
Segmenting axon and myelin from microscopic images is relevant for studying the peripheral and central nervous system and for validating new MRI techniques that aim at quantifying tissue microstructure. While several software packages have been proposed, their interface is sometimes limited and/or they are designed to work with a specific modality (e.g., scanning electron microscopy only). Here we introduce AxonSeg, which allows to perform automatic axon and myelin segmentation on histology images, and to extract relevant morphometric information, such as axon diameter distribution, axon density and the myelin g-ratio. AxonSeg includes a simple and intuitive MATLAB-based graphical user interface and can easily be adapted to a variety of imaging modalities. The main steps of AxonSeg consist of: (i) image pre-processing, (ii) pre-segmentation of axons over a cropped image and discriminant analysis to select the best parameters based on axon shape and intensity information, (iii) automatic axon and myelin segmentation over the full image and (iv) atlas-based statistics to extract morphometric information. Segmentation results from standard optical microscopy (OM), scanning electron microscopy (SEM) and coherent anti-Stokes Raman scattering (CARS) microscopy are presented, along with validation against manual segmentations. Being fully-automatic after a quick manual intervention on a cropped image, we believe AxonSeg will be useful to researchers interested in large throughput histology. AxonSeg is open source and freely available at: https://github.com/neuropoly/axonseg
Molecular order imaging in lipid membranes by polarimetric two-photon fluorescence microscopy
Probing molecular disorder in lipid membranes by two photon fluorescence polarimetry
International audienc