Landmark-based retrieval of inflamed skin vessels enabled by 3D correlative intravital light and volume electron microscopy

The nanometer spatial resolution of electron microscopy imaging remains an advantage over light microscopy, but the restricted field of view that can be inspected and the inability to visualize dynamic cellular events are definitely drawbacks of standard transmission electron microscopy (TEM). Sever...

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Bibliographic Details
Published in:Histochemistry and cell biology 2022-08, Vol.158 (2), p.127-136
Main Authors: Mildner, Karina, Breitsprecher, Leonhard, Currie, Silke M., Stegmeyer, Rebekka I., Stasch, Malte, Volkery, Stefan, Psathaki, Olympia Ekaterini, Vestweber, Dietmar, Zeuschner, Dagmar
Format: Article
Language:English
Subjects:
Biochemistry
Biomedical and Life Sciences
Biomedicine
Cell Biology
Developmental Biology
Electron microscopes
Image processing
Inflammation
Light microscopy
Microscopy
Original Paper
Scanning electron microscopy
Skin
Spatial discrimination
Transmission electron microscopy
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Summary:The nanometer spatial resolution of electron microscopy imaging remains an advantage over light microscopy, but the restricted field of view that can be inspected and the inability to visualize dynamic cellular events are definitely drawbacks of standard transmission electron microscopy (TEM). Several methods have been developed to overcome these limitations, mainly by correlating the light microscopical image to the electron microscope with correlative light and electron microscopy (CLEM) techniques. Since there is more than one method to obtain the region of interest (ROI), the workflow must be adjusted according to the research question and biological material addressed. Here, we describe in detail the development of a three-dimensional CLEM workflow for mouse skin tissue exposed to an inflammation stimulus and imaged by intravital microscopy (IVM) before fixation. Our aim is to relocate a distinct vessel in the electron microscope, addressing a complex biological question: how do cells interact with each other and the surrounding environment at the ultrastructural level? Retracing the area over several preparation steps did not involve any specific automated instruments but was entirely led by anatomical and artificially introduced landmarks, including blood vessel architecture and carbon-coated grids. Successful retrieval of the ROI by electron microscopy depended on particularly high precision during sample manipulation and extensive documentation. Further modification of the TEM sample preparation protocol for mouse skin tissue even rendered the specimen suitable for serial block-face scanning electron microscopy (SBF-SEM).
ISSN:0948-6143
1432-119X
DOI:10.1007/s00418-022-02119-8