Modern Methods in High-Resolution and Volume Scanning Electron Microscopy Image Segmentation and Double-Sided Immunolabeling for Biomolecular Localization

Mgr. František KITZBERGER, M.Sc.

Disertační práce

Abstract:

This thesis deals with modern high-resolution and volume scanning electron microscopy (HR-SEM and vEM) methods, focusing on double-sided immunolocalization using HR-SEM operating in STEM mode and on vEM data processing, segmentation and visualization. The theoretical part is written as a practical primer, as it summarizes the imaging principles and resolution drivers of HR-SEM, compares major vEM modalities (SBF-SEM, FIB-SEM, and array tomography) outlines the sample preparation and immunolabelling strategies and then describes full analytical pipeline from pre-processing, through manual and automated segmentation approaches to 3D visualization and FAIR data handling. The experimental part comprises three studies, where the theoretical consideration are applied in practice. The first introduces a low-voltage STEM method for possible quantitative double-sided immunogold labelling. The other two apply the vEM workflows o biologically complex tissues, analysing ultrastructural changes in the tick midgut and performing targeted vEM of skin to study early host-pathogen interactions. The thesis treats HR-SEM, vEM, and the data analysis as integrated workflows that shall produce complex but most importantly reproducible and quantitatively interpretable information.

Abstract:

This thesis deals with modern high-resolution and volume scanning electron microscopy (HR-SEM and vEM) methods, focusing on double-sided immunolocalization using HR-SEM operating in STEM mode and on vEM data processing, segmentation and visualization. The theoretical part is written as a practical primer, as it summarizes the imaging principles and resolution drivers of HR-SEM, compares major vEM modalities (SBF-SEM, FIB-SEM, and array tomography) outlines the sample preparation and immunolabelling strategies and then describes full analytical pipeline from pre-processing, through manual and automated segmentation approaches to 3D visualization and FAIR data handling. The experimental part comprises three studies, where the theoretical consideration are applied in practice. The first introduces a low-voltage STEM method for possible quantitative double-sided immunogold labelling. The other two apply the vEM workflows o biologically complex tissues, analysing ultrastructural changes in the tick midgut and performing targeted vEM of skin to study early host-pathogen interactions. The thesis treats HR-SEM, vEM, and the data analysis as integrated workflows that shall produce complex but most importantly reproducible and quantitatively interpretable information.

Keywords

Volume Electron Microscopy, Immunolabelling, High-Resolution Scanning Electron Microscopy, Scanning Transmission Electron Microscopy, Segmentation, BioImage Analysis, Visualization, Rendering, Data Processing

Jazyk práce: angličtina

Datum vytvoření / odevzdání či podání práce: 13. 1. 2025

Obhajoba závěrečné práce

• Vedoucí: Ing. Jana Nebesářová, CSc.

JIHOČESKÁ UNIVERZITA V ČESKÝCH BUDĚJOVICÍCH

Přírodovědecká fakulta

Doktorský studijní program / obor:
Biofyzika / Biofyzika