Department of Biomedical Engineering Posters and Presentations

Title

Big Data Approach to Electron Microscopy Acquisition and Analysis of Neuronal Tissue: Utility in Analysis of Neuronal and Synaptic Alterations in a Mouse Model of 22q11 deletion Syndrome

Document Type

Poster

Keywords

Neuroscience, Imaging, DiGeorge Syndrome

Publication Date

4-2017

Abstract

A novel approach was applied using scanning electron microscopy and block-face imaging to areas of the nuclei of the hypoglossal nerve to compare the neuronal architecture and cellular alterations in the mouse model of DiGeorge Syndrome. Since the swallowing and feeding is frequently compromised in human patients carrying this disease our hypothesis is that XII neurons are affected by the deletion in a way that prevents them to perform their function. Therefore, the focus was on XII cranial nerve, because it contains the neurons innervating the tongue. To accomplish the objective, an approach for data acquisition and analysis is designed that allows sampling the entire area of the XII nucleus at 2nm pixel size. This approach allowed us to apply several strategies for data collection and analysis that are impossible with traditional electron microscopy methods.

Brainstem tissue was collected from wild type mice and LgDel mice (carrying similar 22q11 deletion as the patients with DiGeorge Syndrome) after perfusion with glutaraldehyde. The brainstems containing the XII nucleus were cut into 400 mm sections, fixed with osmium tetroxide, infiltrated with uranium acetate, dehydrated in ascending alcohols, and infiltrated in resin that was polymerized in wafers at 60°C. Thin sections of 100mm were cut with a diamond knife and mounted on silicon wafers for imaging. Imaging was performed using the FEI Helios SEM (FEI), equipped with solid-state circular backscattering detector. Imaging was done using 2kV accelerating voltage and current between 100-400 pAmps. MAPs software was used to integrate at different layers the low magnification data (500x), moderate magnification data (5000x) and high magnification data (80,000x). The images at the highest magnification were taken at 2nm pixel size. Serial tile images from one side of the nucleus were taken and stitched together in one large RAW image, which was further transferred to Photoshop for data conversion. Sections of the large image were then selected and transferred to Arivis software as .tif data for segmentation and analysis. Manual annotations are made and stored using Arivis. The data is exported into a spreadsheet. The purpose of the research is to quantitatively explain cellular degeneration and understand the structural differences between Wild types cells and LgDel cells in hopes of one day treating the issues created by DiGeorge Syndrome.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Open Access

1

Comments

To be presented at GW Annual Research Days 2017.

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Big Data Approach to Electron Microscopy Acquisition and Analysis of Neuronal Tissue: Utility in Analysis of Neuronal and Synaptic Alterations in a Mouse Model of 22q11 deletion Syndrome

A novel approach was applied using scanning electron microscopy and block-face imaging to areas of the nuclei of the hypoglossal nerve to compare the neuronal architecture and cellular alterations in the mouse model of DiGeorge Syndrome. Since the swallowing and feeding is frequently compromised in human patients carrying this disease our hypothesis is that XII neurons are affected by the deletion in a way that prevents them to perform their function. Therefore, the focus was on XII cranial nerve, because it contains the neurons innervating the tongue. To accomplish the objective, an approach for data acquisition and analysis is designed that allows sampling the entire area of the XII nucleus at 2nm pixel size. This approach allowed us to apply several strategies for data collection and analysis that are impossible with traditional electron microscopy methods.

Brainstem tissue was collected from wild type mice and LgDel mice (carrying similar 22q11 deletion as the patients with DiGeorge Syndrome) after perfusion with glutaraldehyde. The brainstems containing the XII nucleus were cut into 400 mm sections, fixed with osmium tetroxide, infiltrated with uranium acetate, dehydrated in ascending alcohols, and infiltrated in resin that was polymerized in wafers at 60°C. Thin sections of 100mm were cut with a diamond knife and mounted on silicon wafers for imaging. Imaging was performed using the FEI Helios SEM (FEI), equipped with solid-state circular backscattering detector. Imaging was done using 2kV accelerating voltage and current between 100-400 pAmps. MAPs software was used to integrate at different layers the low magnification data (500x), moderate magnification data (5000x) and high magnification data (80,000x). The images at the highest magnification were taken at 2nm pixel size. Serial tile images from one side of the nucleus were taken and stitched together in one large RAW image, which was further transferred to Photoshop for data conversion. Sections of the large image were then selected and transferred to Arivis software as .tif data for segmentation and analysis. Manual annotations are made and stored using Arivis. The data is exported into a spreadsheet. The purpose of the research is to quantitatively explain cellular degeneration and understand the structural differences between Wild types cells and LgDel cells in hopes of one day treating the issues created by DiGeorge Syndrome.