Cellular structure can be imaged at nanometer resolution using electron microscopy, but the biological molecules remain invisible in grayscale electron micrographs. With Correlative Light and Electron Microscopy (CLEM), fluorescence recorded on a separate light microscope can be used to identify molecules in colour, but the resolution gap with electron microscope precludes accurate localization. The use of super-resolution fluorescence microscopy techniques combined with CLEM[2,3] holds great promise but is still one order of magnitude off in resolution compared to EM. In addition, accurate registration of the separate images may be challenging and correlation may require expert procedures to maintain, e.g., protein photo-switching under EM preparation conditions. Thus, while CLEM adds information represented in the colors of visible light, molecular identification and localization at the resolution of the electron microscope is still hampered. Therefore, we are exploring novel approaches to achieve color-EM using the electron beam to add information about the specimen to the electron micrographs.
Detection of electron-beam excited luminescence has been explored in the past. Cross-sections for visible luminescence (also called cathodoluminescence) are, however, small and most biological fluorescent labels are destroyed before sufficient signal has been collected to allow high-resolution localization . Phosphorescent nanoparticles are considered as non-bleaching alternative, but particle sizes are not yet well-defined and bio-functionalization has in most cases still to be pursued.
Here, we explore detection of X-ray fluorescence to identify nanoparticle labels at EM resolution in colour on tissue. We use Au and CdSe colloidal quantum dots, immuno-targeted to guanine quadruplexes, resp. insulin, as molecular labels in large-scale electron microscopy of pancreatic tissue. Energy dispersive detection of X-ray emission (EDX) during electron irradiation allows discrimination of both nanoparticles based on their elemental (Au vs Cd) composition. We will present electron microscopy images overlaid with false colour elemental maps displaying single nanoparticles. Separate resolution tests have shown that individual Au particles down to 3 nm in size can be detected using EDX, highlighting the potential for coloured identification of materials in biological electron microscopy, revealing both compositional and ultrastructural information at nanometer-scale resolution.
 P. de Boer, J. P. Hoogenboom, B. N. G. Giepmans, Nature Methods 12, 503 (2015)
 Kopek, B.G., et al., Proc. Natl. Acad. Sci. U. S. A. 109, 6136 (2012)
 Johnson, E. et al., Scientific Reports 5 (2015)
 Niitsuma, J., Oikawa, H., Kimura, E., Ushiki, T. & Sekiguchi, T., J. Electron Microscopy 54, 325 (2005).
 Glenn, D.R. et al., Scientific Reports 2 (2012)
 A. Loukanov, N. Kamasawa, R. Danev, R. Shigemoto, K. Nagayama, Ultramicroscopy 110,366 (2010)
To cite this abstract:Marijke Scotuzzi, Jeroen Kuipers, Dasha I. Wensveen, Pascal de Boer, Cornelis W. Hagen, Ben N G Giepmans, Jacob P. Hoogenboom; High-resolution identification of immuno-labelling nanoparticles on tissue using X-ray detection. The 16th European Microscopy Congress, Lyon, France. https://emc-proceedings.com/abstract/high-resolution-identification-of-immuno-labelling-nanoparticles-on-tissue-using-x-ray-detection/. Accessed: December 1, 2022
EMC Abstracts - https://emc-proceedings.com/abstract/high-resolution-identification-of-immuno-labelling-nanoparticles-on-tissue-using-x-ray-detection/