Label-free 3D-CLEM Using Endogenous Tissue Landmarks
Emerging 3D correlative light and electron microscopy approaches enable studying neuronal structure-function relations at unprecedented depth and precision. However, established protocols for the correlation of light and electron micrographs rely on the introduction of artificial fiducial markers, s...
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Veröffentlicht in: | iScience 2018-08, Vol.6, p.92-101 |
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Format: | Artikel |
Sprache: | eng |
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Zusammenfassung: | Emerging 3D correlative light and electron microscopy approaches enable studying neuronal structure-function relations at unprecedented depth and precision. However, established protocols for the correlation of light and electron micrographs rely on the introduction of artificial fiducial markers, such as polymer beads or near-infrared brandings, which might obscure or even damage the structure under investigation. Here, we report a general applicable “flat embedding” preparation, enabling high-precision overlay of light and scanning electron micrographs, using exclusively endogenous landmarks in the brain: blood vessels, nuclei, and myelinated axons. Furthermore, we demonstrate feasibility of the workflow by combining in vivo 2-photon microscopy and focused ion beam scanning electron microscopy to dissect the role of astrocytic coverage in the persistence of dendritic spines.
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•Flat embedding of vibratome sections for precise correlation of LM and SEM•Endogenous landmarks are ideal fiducials for CLEM•The glass slide serves as an absolute reference for precise alignment in yz•Correlation of perisynaptic ensheathment and dendritic spine lifetime
Neuroscience; Techniques in Neuroscience; Biological Sciences Research Methodologies; Biological Sciences Tools |
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ISSN: | 2589-0042 2589-0042 |
DOI: | 10.1016/j.isci.2018.07.012 |