title: Bridging microscopes: 3D correlative light and scanning electron microscopy of complex biological structures.
abstract: The rationale of correlative light and electron microscopy (CLEM) is to collect data on different information levels--ideally from an identical area on the same sample--with the aim of combining datasets at different levels of resolution to achieve a more holistic view of the hierarchical structural organization of cells and tissues. Modern three-dimensional (3D) imaging techniques in light and electron microscopy opened up new possibilities to expand morphological studies into the third dimension at the nanometer scale and over various volume dimensions. Here, we present two alternative approaches to correlate 3D light microscopy (LM) data with scanning electron microscopy (SEM) volume data. An adapted sample preparation method based on high-pressure freezing for structure preservation, followed by freeze-substitution for multimodal en-bloc imaging or serial-section imaging is described. The advantages and potential applications are exemplarily shown on various biological samples, such as cells, individual organisms, human tissue, as well as plant tissue. The two CLEM approaches presented here are per se not mutually exclusive, but have their distinct advantages. Confocal laser scanning microscopy (CLSM) and focused ion beam-SEM (FIB-SEM) is most suitable for targeted 3D correlation of small volumes, whereas serial-section LM and SEM imaging has its strength in large-area or -volume screening and correlation. The second method can be combined with immunocytochemical methods. Both methods, however, have the potential to extract statistically relevant data of structural details for systems biology.
authors.affiliation: Electron Microscopy ETH Zurich – EMEZ, ETH Zurich, Switzerland.
authors.forename: Miriam S
authors.initials: MS
authors.lastname: Lucas
authors.forename: Maja
authors.initials: M
authors.lastname: Günthert
authors.forename: Philippe
authors.initials: P
authors.lastname: Gasser
authors.forename: Falk
authors.initials: F
authors.lastname: Lucas
authors.forename: Roger
authors.initials: R
authors.lastname: Wepf
mesh_terms: D000818:Animals; D020369:Bradyrhizobium / Q000648:ultrastructure; D017173:Caenorhabditis elegans / Q000648:ultrastructure; D002478:Cells, Cultured; D015925:Cryopreservation; D055032:Electron Microscope Tomography; D004817:Epidermis / Q000378:metabolism / Q000648:ultrastructure; D007887:Fabaceae / Q000382:microbiology / Q000648:ultrastructure; D005963:Glucosylceramides / Q000378:metabolism; D006801:Humans; D021621:Imaging, Three-Dimensional; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D008867:Microtomy; D016613:Plastic Embedding; D012867:Skin / Q000648:ultrastructure; D016707:Tissue Fixation
chemical_list: D005963:Glucosylceramides