Cellular complexity is unraveled at nanometer resolution using electron microscopy (EM), but interpretation of macromolecular functionality is hampered by the difficulty in interpreting grey-scale images and the unidentified molecular content. We perform large-scale EM on mammalian tissue complemented with energy-dispersive X-ray analysis (EDX) to allow EM-data analysis based on elemental composition. Endogenous elements, labels (gold and cadmium-based nanoparticles) as well as stains are analyzed at ultrastructural resolution. This provides a wide palette of colors to paint the traditional grey-scale EM images for composition-based interpretation. Our proof-of-principle application of EM-EDX reveals that endocrine and exocrine vesicles exist in single cells in Islets of Langerhans. This highlights how elemental mapping reveals unbiased biomedical relevant information. Broad application of EM-EDX will further allow experimental analysis on large-scale tissue using endogenous elements, multiple stains, and multiple markers and thus brings nanometer-scale ‘color-EM’ as a promising tool to unravel molecular (de)regulation in biomedicine.

Electron microscopy (EM) is a powerful tool to study cellular complexity at nanometer resolution. Traditionally, EM images are acquired from small fields of view, but several new techniques allow routine acquisition of large 2D and 3D image stacks. However, in the resulting vast amount of greyscale data interpretation in terms of macromolecular functionality is difficult. With Correlative Light and Electron Microscopy (CLEM), fluorescence can be used to identify molecules in color[1] but the resolution gap with EM precludes accurate localization. The use of superresolution fluorescence microscopy techniques combined with CLEM[2,3] holds great promise but may be hard to achieve and the color palette limited. We are exploring novel approaches to achieve color-EM using the electron beam to add information about the specimen to the electron micrographs.Large-scale EM is complemented with energy-dispersive-X-ray analysis (EDX) to allow EM analysis based on elemental composition. Different endogenous elements as well as exogenously introduced elements, including labels (gold-and cadmium-based nanoparticles) and stains, can be discriminated at ultrastructure resolution. This provides a wide palette of colors to paint the traditional grey-scale EM images for composition-based interpretation. Our proof-of-principle application of EM-EDX reveals that endocrine and exocrine vesicles exists in single cells in a Type-1 diabetes rat model (see Figure 1), highlighting how elemental mapping provides biomedical relevant information without bias. Broad application of EM-EDX will further allow experimental analysis on different kind of tissues, stains, and labels and thus brings nanometer-scale 'color-EM' as a promising tool to unravel molecular (de)regulation in biomedicine.

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)[1], 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 [4]. 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[5]. Here, we explore detection of X‐ray fluorescence to identify nanoparticle labels[6] 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.