A. Srinivasa Raja
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1
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....
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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....
Journal article
(2021)
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Aditi Srinivasa Raja, Pascal de Boer, Ben N.G. Giepmans, Jacob P. Hoogenboom
Electron microscopy is crucial for imaging biological ultrastructure at nanometer resolution. However, electron irradiation also causes specimen damage, reflected in structural and chemical changes that can give rise to alternative signals. Here, luminescence induced by electron-beam irradiation is reported across a range of materials widely used in biological electron microscopy. Electron-induced luminescence is spectrally characterized in two epoxy (Epon, Durcupan) and one methacrylate resin (HM20) over a broad electron fluence range, from 10−4 to 103 mC cm−2, both with and without embedded biological samples. Electron-induced luminescence is pervasive in polymer resins, embedded biomaterial, and occurs even in fixed, whole cells in the absence of resin. Across media, similar patterns of intensity rise, spectral red-shifting, and bleaching upon increasing electron fluence are observed. Increased landing energies cause reduced scattering in the specimen shifting the luminescence profiles to higher fluences. Predictable and tunable electron-induced luminescence in natural and synthetic polymer media is advantageous for turning many polymers into luminescent nanostructures or to fluorescently visualize (micro)plastics. Furthermore, these findings provide perspective to direct electron-beam excitation approaches like cathodoluminescence that may be obscured by these nonspecific electron-induced signals.
...
Electron microscopy is crucial for imaging biological ultrastructure at nanometer resolution. However, electron irradiation also causes specimen damage, reflected in structural and chemical changes that can give rise to alternative signals. Here, luminescence induced by electron-beam irradiation is reported across a range of materials widely used in biological electron microscopy. Electron-induced luminescence is spectrally characterized in two epoxy (Epon, Durcupan) and one methacrylate resin (HM20) over a broad electron fluence range, from 10−4 to 103 mC cm−2, both with and without embedded biological samples. Electron-induced luminescence is pervasive in polymer resins, embedded biomaterial, and occurs even in fixed, whole cells in the absence of resin. Across media, similar patterns of intensity rise, spectral red-shifting, and bleaching upon increasing electron fluence are observed. Increased landing energies cause reduced scattering in the specimen shifting the luminescence profiles to higher fluences. Predictable and tunable electron-induced luminescence in natural and synthetic polymer media is advantageous for turning many polymers into luminescent nanostructures or to fluorescently visualize (micro)plastics. Furthermore, these findings provide perspective to direct electron-beam excitation approaches like cathodoluminescence that may be obscured by these nonspecific electron-induced signals.
In the past decades, correlative light and electron microscopy (CLEM) methods have evolved from being mostly used by a few pioneering, specialist labs to a collection of techniques and workflows practiced by a broad group of researchers in structural biology. In most cases, CLEM involves a distinct set of sequentially used specimen preparation and labeling techniques, followed by diverse types of light and electron microscopy techniques. This chapter focuses on those areas in present-day CLEM that are faced with challenges for which these advantages of integrated microscopes may well be key for further advancement. These areas are large-scale and high-throughput correlated (volume) microscopy, super-resolution localization in resin or cryo-frozen sections, fluorescence-guided focused ion beam milling for cryo-electron tomography, and the integration of sample preparation and transfer. Ultimately this should lead to the development of specific integrated CLEM systems with complete and fully automated workflows, leading to high-throughput and high-yield systems.
...
In the past decades, correlative light and electron microscopy (CLEM) methods have evolved from being mostly used by a few pioneering, specialist labs to a collection of techniques and workflows practiced by a broad group of researchers in structural biology. In most cases, CLEM involves a distinct set of sequentially used specimen preparation and labeling techniques, followed by diverse types of light and electron microscopy techniques. This chapter focuses on those areas in present-day CLEM that are faced with challenges for which these advantages of integrated microscopes may well be key for further advancement. These areas are large-scale and high-throughput correlated (volume) microscopy, super-resolution localization in resin or cryo-frozen sections, fluorescence-guided focused ion beam milling for cryo-electron tomography, and the integration of sample preparation and transfer. Ultimately this should lead to the development of specific integrated CLEM systems with complete and fully automated workflows, leading to high-throughput and high-yield systems.