Correlative microscopy incorporates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Several approaches exist for correlative microscopy, most of which have used the green fluorescent protein (GFP) as the label for light microscopy. Here we use chemical tagging and synthetic fluorophores instead, in order to achieve protein-specific labeling, and to perform multicolor imaging. We show that synthetic fluorophores preserve their post-embedding fluorescence in the presence of uranyl acetate. Post-embedding fluorescence is of such quality that the specimen can be prepared with identical protocols for scanning electron microscopy (SEM) and transmission electron microscopy (TEM); this is particularly valuable when singular or otherwise difficult samples are examined. We show that synthetic fluorophores give bright, well-resolved signals in super-resolution light microscopy, enabling us to superimpose light microscopic images with a precision of up to 25 nm in the x–y plane on electron micrographs. To exemplify the preservation quality of our new method we visualize the molecular arrangement of cadherins in adherens junctions of mouse epithelial cells.
Journal of Structural Biology
Perkovic M, Kunz M, Endesfelder U, et al. Correlative light- and electron microscopy with chemical tags. Journal of Structural Biology. 2014;186(2):205-213. doi:10.1016/j.jsb.2014.03.018
Perkovic, M., Kunz, M., Endesfelder, U., Bunse, S., Wigge, C., Yu, Z., … Frangakis, A. S. (2014). Correlative light- and electron microscopy with chemical tags. Journal of Structural Biology. Elsevier. https://doi.org/10.1016/j.jsb.2014.03.018
Perkovic, Mario, Michael Kunz, Ulrike Endesfelder, Stefanie Bunse, Christoph Wigge, Zhou Yu, Victor-Valentin Hodirnau, et al. “Correlative Light- and Electron Microscopy with Chemical Tags.” Journal of Structural Biology. Elsevier, 2014. https://doi.org/10.1016/j.jsb.2014.03.018.
M. Perkovic et al., “Correlative light- and electron microscopy with chemical tags,” Journal of Structural Biology, vol. 186, no. 2. Elsevier, pp. 205–213, 2014.
Perkovic M, Kunz M, Endesfelder U, Bunse S, Wigge C, Yu Z, Hodirnau V-V, Scheffer MP, Seybert A, Malkusch S, Schuman EM, Heilemann M, Frangakis AS. 2014. Correlative light- and electron microscopy with chemical tags. Journal of Structural Biology. 186(2), 205–213.
Perkovic, Mario, et al. “Correlative Light- and Electron Microscopy with Chemical Tags.” Journal of Structural Biology, vol. 186, no. 2, Elsevier, 2014, pp. 205–13, doi:10.1016/j.jsb.2014.03.018.
All files available under the following license(s):
Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported (CC BY-NC-ND 3.0):