Imaging green fluorescent protein-labeled neurons using light and electron microscopy

Graham Knott
2013
Article

Résumé

The ability to observe axons and dendrites with transmission electron microscopy (EM) after they have been previously imaged live with laser-scanning microscopy is a useful technique to study their synaptic connectivity. This protocol provides a detailed method by which neurons that were imaged in a live brain or slice culture can be reimaged using EM. First, brain tissue expressing green fluorescent protein (GFP) is chemically fixed. Then, an immunocytochemistry process is used to render the fluorescent protein electron dense so that it can first be located using light microscopy and then serial thin-sectioned for EM so that the ultrastructure of specific parts of neurites can be analyzed in three dimensions. Patterns of blood vessels observed in the live brain are used to locate the previously imaged neurons. The method described here allows for a complete three-dimensional (3D) reconstruction to be made of the imaged structures from serial electron micrographs.

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https://infoscience.epfl.ch/record/188449?ln=fr

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A protocol for preparing GFP-labeled neurons previously imaged in vivo and in slice preparations for light and electron microscopic analysis

Graham Knott, Egbert Welker

In vivo imaging of green fluorescent protein (GFP)-labeled neurons in the intact brain is being used increasingly to study neuronal plasticity. However, interpreting the observed changes as modifications in neuronal connectivity needs information about synapses. We show here that axons and dendrites of GFP-labeled neurons imaged previously in the live mouse or in slice preparations using 2-photon laser microscopy can be analyzed using light and electron microscopy, allowing morphological reconstruction of the synapses both on the imaged neurons, as well as those in the surrounding neuropil. We describe how, over a 2-day period, the imaged tissue is fixed, sliced and immuno-labeled to localize the neurons of interest. Once embedded in epoxy resin, the entire neuron can then be drawn in three dimensions (3D) for detailed morphological analysis using light microscopy. Specific dendrites and axons can be further serially thin sectioned, imaged in the electron microscope (EM) and then the ultrastructure analyzed on the serial images.

2009

Chargement

Biomechanics of Vascular Wall

Rana Saitta-Rezakhaniha

This thesis contributes to the field of biomechanics of vascular wall. The focus is particularly on the microstructure of vascular elastin and collagen constituents and their contribution to the macroscopic mechanical behavior of the wall. The analysis is done in the framework of continuum mechanics. The work characterizes structural features of elastin and collagen fibers using microscopy techniques and introduces these features to constituent-based constitutive models. The models are applied to the experimental data, derived from inflation-extension tests, to predict the gross mechanical behavior of the tissue. The developed constitutive models could be further used to study in detail the mechanics of vascular tissue in health and disease. The thesis is presented in form of an introduction, four chapters (corresponding to four papers) and a conclusion. The introduction provides the motivation for this thesis as well as the background on vascular wall structure and mechanics. A brief description of the imaging techniques used is presented. Also, structural constitutive modeling of vascular wall is briefly discussed with particular attention to the modeling of elastin and collagen constituents. The first paper focuses on anisotropic properties of elastin in veins. We show that earlier constituent-based strain energy functions (SEFs), where elastin is modeled as an isotropic material, fail in describing accurately the tissue response to inflation-extension loading. We hypothesize that these shortcomings are partly due to unaccounted anisotropic properties of elastin. We extend the previously developed biomechanical model in our laboratory (Zulliger et al., 2004, J. Biomech., 37(7): 989-1000 (2004)) to account for elastin anisotropy and present an anisotropic strain energy function for elastin with one family of fibers in the longitudinal direction. The model is validated using experimental data from inflation-extension tests on rabbit facial veins. The tissue is tested under a fully relaxed smooth muscle state, for longitudinal stretch ratios ranging from 100% to 130% of the in vivo length. The model with the anisotropic elastin fits well the data for a wide range of longitudinal stretch ratios. The main finding of this paper is that the anisotropic description of elastin is required for a full 3-D characterization of the biomechanics of the venous wall. The second paper addresses the role of elastin in anisotropic properties of arteries with particular attention to the structural organization of elastin. A constituent-based model including an anisotropic elastin, with one family of fibers in the circumferential direction, is presented. Micro-structural imaging, based on electron microscopy techniques, is used to support this anisotropy. Inflationˆextension tests, on intact and elastase-treated arteries, provide a data set to validate the model and to study the effect of elastin removal. We show that the SEF, with an anisotropic elastin part, characterizes more accurately the mechanical properties of the arterial wall as compared to models with simply an isotropic elastin. Transmission electron microscopy (TEM) and serial block-face scanning electron microscopy (SBF-SEM) show interlamellar elastin fibers in the circumferential direction and therefore support the nature of the assumed anisotropy. The model predicts an earlier engagement of collagen in elastase-treated arteries compared to the intact arteries and thus suggests a clear functional interaction between the elastin and collagen component that is often neglected in constituent-based SEFs. The third paper presents a structural constitutive model of the vascular wall which integrates both waviness and orientational dispersion of collagen fibers. We extend the model of Zulliger et al., which already accounts for collagen waviness, to include orientational distribution of collagen. We study the effect of parameters related to the orientational distribution on macro-mechanical behavior of the tissue during inflation-extension tests. The model is further applied to the experimental data from rabbit facial veins. The model accurately fits the experimental data of veins, but it does not improve the quality of the fit compared to the one without dispersion. We show that the orientational dispersion of collagen fibers can be compensated by a less abrupt and shifted to higher strain collagen engagement pattern. This should be taken into consideration when the model is used to fit experimental data and model parameters are used to study structural modifications of collagen fiber network in physiology and disease. In the fourth paper, we measure and quantify the waviness and orientational distributions of collagen in rabbit carotids. 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In addition, our measurements reveal four axially symmetric families of fibers with mean orientations of 0°, 90°, 43° and -43° and circular standard deviations of 40°, 47°, 37° and 37°, with respect to the axial direction, respectively. To the best of our knowledge, this is the first study focusing on structural properties of collagen in the zero stress state and quantifying fiber waviness. The results of this study can be used to develop more precise structural models of the adventitia including waviness and orientational dispersion of fibers. The conclusion section summarizes the main results of the thesis, presents the improvements made over previous studies, and proposes future perspectives of this work.

EPFL2010

Chargement

Electron microscopy offers a wide range of techniques to study materials. Environmental Scanning Electron Microscopy (ESEM), in particular, allows the characterisation of samples at high-temperature using a laser heating stage, and under various gaseous atmospheres leading to direct observation of the sample surface morphology dynamics in conditions that are close to those in real operating devices (i.e. at high temperature in various gases). Solid Oxide Fuel Cells (SOFC) operating around 800°C in both oxidising and reducing gases, with proven co-generation efficiencies of 90%, offer very promising solutions in the ongoing energy transition. However, production costs and materials degradation in those demanding operating conditions remain significant barriers to their large-scale deployment. Studying SOFC materials' degradation through a combination of ESEM with other advanced microscopy techniques fulfilled the double aim of demonstrating the power of an ESEM and improving the understanding of SOFC materials' degradation during operation. Two materials from the SOFC stack were selected for degradation case studies at around 800°C in oxidising/reducing gases: coated ferritic steel interconnect and Ni-YSZ anode. The objectives were to explore the parameters' influence (temperature, heating ramp, pressure, gas exposure...), set the boundaries and define the guidelines of the ESEM technique while demonstrating its capabilities. The two different materials sets were selected for their relevance both for the SOFC field and for their different specific characteristics allowing a wide range of experiments to be explored. A specific method for the study of the oxidation of coated steel was developed combining ESEM, Focused Ion Beam (FIB) and Scanning Transmission Electron Microscopy (STEM). Post-test segmentation of the in-situ observed images demonstrated the possibility to obtain quantitative data. FIB sample preparation from the exact site of ESEM observation allowed to correlate the underlying microstructure with the in-situ surface observation. Applying this method, oxidation mechanisms of several coatings were characterised and specific behaviours observed and explained such as the appearance of oxide ridges and the behaviour of a cerium barrier layer in case of cobalt-cerium coated steel. The heating ramp speed and the oxidation temperature were demonstrated to be crucial parameters to obtain the desired microstructure. The nitriding pretreatment of interconnect was investigated too and shows promising results for improving the steel oxidation resistance. Another combination of techniques was developed for the study of the Ni-YSZ anode by complementing the ESEM observation with continuous Mass Spectrometer (MS) acquisition. The first reduction of the NiO-YsZ anode was analysed under gas flows of hydrogen and hydrocarbons. Redox cycling effect on the anode microstructure was investigated and the resulting observations were in line with models from the literature for the reduction and re-oxidation kinetics. Finally, the demonstration of an active solid oxide cell in the microscope operating at more than 700°C under mixed gas paves the way to further possible studies approaching the device's observation in real operating conditions. Limitation of the ESEM techniques are discussed and recommendations for further improvements and perspectives given.

EPFL2020

Chargement

EPFL2020

Biomechanics of Vascular Wall

Rana Saitta-Rezakhaniha

EPFL2010