title: Immuno correlative light and electron microscopy on Tokuyasu cryosections.
abstract: Finding a rare structure by electron microscopy is the equivalent of finding a "needle in a haystack." Correlative light- and immunoelectron microscopy (CLEM) on Tokuyasu cryosections is a sophisticated technique to address this challenge. Hereby, fluorescently labeled structures of interest are identified in an overview image by light microscopy and subsequently traced in electron microscopy. While the direct transfer and imaging of the same sections from optical to electron microscopy enables straightforward correlation, the sample preparation is crucial and technically demanding. We provide a detailed guide outlining the critical steps for sample embedding, cryosectioning, immunolabeling, and imaging. In the example provided, we use CLEM to trace aggregates formed in a zebrafish myopathy model expressing enhanced green fluorescent protein (eGFP) tagged actin. In our case, only a few muscle fibers express eGFP-actin with a subset of fibers containing aggregates. By fluorescence microscopy, we are able to identify the aggregates in the zebrafish tissue, and we subsequently, use immunoelectron microscopy to image the same structures at high resolution. The CLEM method described here using Tokuyasu cryosections can be applied to a large range of samples including small organisms, tissue samples, and cells.
authors.affiliation: Monash Micro Imaging, Monash University, Melbourne, Victoria, Australia.
authors.forename: Viola M J
authors.initials: VM
authors.lastname: Oorschot
authors.affiliation: School of Biological Sciences, Faculty of Sciences, Monash University, Melbourne, Victoria, Australia.
authors.forename: Tamar E
authors.initials: TE
authors.lastname: Sztal
authors.affiliation: School of Biological Sciences, Faculty of Sciences, Monash University, Melbourne, Victoria, Australia.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Bryson-Richardson
authors.affiliation: Monash Micro Imaging, Monash University, Melbourne, Victoria, Australia; Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, Australia.
authors.forename: Georg
authors.initials: G
authors.lastname: Ramm
mesh_terms: D000818:Animals; D015925:Cryopreservation; D057918:Fiducial Markers; D005629:Frozen Sections; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D007091:Image Processing, Computer-Assisted; D046529:Microscopy, Electron, Transmission / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D016253:Microscopy, Immunoelectron / Q000379:methods; D018482:Muscle, Skeletal / Q000378:metabolism / Q000648:ultrastructure*; D016610:Tissue Embedding; D016707:Tissue Fixation; D015027:Zebrafish
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: CLEM; GFP; Immuno-EM; Tokuyasu; Zebrafish