Electron Beam-Induced Fluorescence Localization : Implementation and Feasibility in Integrated Light-Electron Microscopy

Correlative light-electron microscopy (CLEM) combines the molecular specificity of fluorescence microscopy (FM) with the ultrastructural resolution of electron microscopy (EM) to provide functional information in the context of structural detail. However, the correlation between the two modalities is hindered by a 100-fold resolution gap. Superresolution fluorescence microscopy (SR-FM) has enabled more accurate correlation in CLEM, aided by advancements in sample preparation, bimodal registration, and optimized workflows. As the resolution of FM approaches that of EM, SR-FM becomes increasingly challenging due to the need for accurate registration and preservation of fluorophore properties during EM sample preparation. Integrated microscopes can remove the need for external alignment markers and achieve high registration accuracies, eliminating sample deformations and facilitating SR-CLEM. Yet, this necessitates samples that are simultaneously amenable to both FM and EM. While advancements are being made to engineer fixation-resistant fluorescent proteins and develop preparation protocols for preserving in-resin fluorescence, it is worth exploring the possibilities of the unified platform that integrated light-electron microscopy offers, especially for SR-FM. In caseswhere traditional SR-FM cannot be used due to vacuum or other limitations, the combination of both light and electron microscopy can provide valuable multi-modal information. It also enables central control of the experimental system, thereby offering new ways to manipulate, process, and interpret fluorescence data. This thesis aims to investigate and utilize luorescent response to electron irradiation using integrated light-electronmicroscopy....