Fluorescent labeling of resin-embedded sections for correlative electron microscopy using tomography-based contrast enhancement.
Locating areas of interest by electron microscopy can be laborious. This is particularly true for electron tomography, where the use of thicker sections may obscure relevant details in the projection images. We evaluated the applicability of fluorescent probes to thin plastic sections, in combination with fluorescence microscopy, as an aid in selecting areas for subsequent electron microscopic analysis. We show that pre-embedding labeling of DNA and RNA with acridine orange yielded a predominant nuclear stain. The stain greatly reduced the time needed to scan sections for mitotic cells, or cells with characteristic nuclei such as neutrophils. Post-embedding labeling with SYTOX green yielded a nuclear stain comparable to acridine orange, and wheat germ agglutinin (WGA) conjugated to Alexa Fluor 488 labeled mucous granules and the Golgi area in intestinal goblet cells. The fluorescent labels were visualized directly on sections on electron microscope grids. It was therefore possible to establish a coordinate system based on the position of the grid bars, allowing for easy retrieval of selected areas. Because the fluorescent probes were incompatible with osmium tetroxide treatment, contrast in the sections was faint. We propose a simplified electron tomography procedure for the generation of 2D views with enhanced contrast and resolution.
van Driel
Linda F
LF
Department of Molecular Cell Biology, Section Electron Microscopy, Leiden University Medical Center, Leiden, The Netherlands. L.van_Driel@lumc.nl
Knoops
Kèvin
K
Koster
Abraham J
AJ
Valentijn
Jack A
JA
D003287:Contrast Media; D005456:Fluorescent Dyes; D008854:Microscopy, Electron / Q000379:methods*; D008856:Microscopy, Fluorescence; D009696:Nucleic Acids / Q000378:metabolism*; D016610:Tissue Embedding / Q000379:methods*; D014054:Tomography / Q000379:methods*
D003287:Contrast Media; D005456:Fluorescent Dyes; D009696:Nucleic Acids