EDX Imaging: Electron Microscopy beyond the grey-scale

Electron Microscopy holds the key to understanding the ultrastructure of biological systems, but finding what you are looking for in the high resolution EM data can be challenging. In a method known as energy dispersive X-ray analysis (EDX), Transmission Electron Microscopy is complemented by an X-ray detector which reveals the elemental composition of a sample on the nanometer scale. Researchers can use this approach to display organelle-specific spectra, as Ben Giepmans, member of the Correlative Light Microscopy Dutch Flagship Node explains. In this article, Ben Giepmans shares excerpts from Pirozzi et al, 2018, “ColorEM: analytical electron microscopy for element-guided identification and imaging of the building blocks of life,” and highlights studies done in collaboration with his lab to explain the EDX approach. EDX is currently available in open access as part of Euro-BioImaging’s Proof-of-Concept Study.

This article features excerpts from the paper: Pirozzi, N.M., Hoogenboom, J.P. & Giepmans, B.N.G. ColorEM: analytical electron microscopy for element-guided identification and imaging of the building blocks of life. Histochem Cell Biol 150, 509–520 (2018). https://doi.org/10.1007/s00418-018-1707-4. PMID: 30120552; PMCID: PMC6182685. Used under a Creative Commons Attribution 4.0 international license. (http://creativecommons.org/licenses/by/4.0/>)

To read the full article, click here

We are today talking about EDX imaging. Please provide a short summary of this type of imaging:

Modified excerpt from Pirozzi et al., 2018, used under Creative Commons Attribution 4.0 international license (http://creativecommons.org/licenses/by/4.0/):

”EDX is an elemental composition analysis technique that can be easily performed on any existing scanning (transmission) EM with the addition of an X-ray detector. EDX utilizes specific radiation produced when the incident beam of the electron microscope creates an electron vacancy in the sample (Fig. 1a). At the atomic level, electron vacancies created from the incident electron beam are quickly filled by electrons of higher energy shells, releasing radiation of the difference in electron binding energies in the form of characteristic X-rays. These characteristic X-rays are energy-specific to the atom from which they were produced and thus reveal the elemental composition of a sample (...) For a comprehensive background on X-ray principles, hardware and analysis for non-physicists we highly recommend the book by Friel et al. (2017).”

What are some applications of EDX?

Modified excerpt from Pirozzi et al., 2018, used under Creative Commons Attribution 4.0 international license (http://creativecommons.org/licenses/by/4.0/):

”EDX has been widely applied in geology and material science since the 1940s. The transition to life sciences has been marked by developments in X-ray detector technology. The first applications came in the 1970s after the introduction of energy-dispersive semiconductor-based detectors (Echlin 1971; Roomans and Von Euler 1996). New advances with silicon drift X-ray detectors make it possible for EDX to bridge to life sciences as lighter elements more relevant in biological samples can now be measured with greater signal to noise ratios, and achieved theoretical resolution, independent of temperature and count rates (Friel et al. 2017). The next generation of detectors are annular, surrounding the sample and increasing the solid angle which increases count rate and shortens acquisition time (Teng et al. 2018).

ColorEM of endogenous elements in biological samples, such as the phosphorus enriched in membranes and DNA, nitrogen in polypeptides, and sulfur in methionine-rich and cysteine-rich proteins can be qualitatively measured, mapped, and overlayed on EM images of these samples using EDX. The additional dimension of the electron image aids identification, making analysis more objective and less interpretation-based.

Additionally, EDX can be used in immuno-EM, where different particles are applied as EM immunolabels and then differentiated on the basis of their elemental content, as seen with gold nanoparticles and quantum dots with a cadmium selenide core. Quantification of biological samples presents new challenges where even the estimation of the background signal required new considerations, stimulating new modeling (Roomans and Kuypers 1980). Thus, in life sciences, EDX progressed from whole cell spectra to organelle-specific spectra (Somlyo et al. 1977b), to full mapping with individual spectra collected in nm-scale pixels (Scotuzzi et al. 2017).”

Tell us a bit more about a specific project that was done in your facility using this technology? What scientific questions were you addressing?

Ben: In a study with Scotuzzi et al., 2017, it was demonstrated that EDX can be a promising way to look at molecular (de)regulation in biomedicine. This study used large-scale EM on mammalian tissue complemented with EDX to identify cells, organelles and molecules and their elemental composition. We also used EDX in the human ‘nPOD’ biobank (details: nPOD.org) to reveal the identity of granules in cells of the pancreas, showing that some cell types contain both hormones and exocrine granules. If this unexpected finding is related to the ultimate destruction of beta cells is currently under investigation. We do now have two microscopes equipped with EDX detectors, and many users of the facility apply EDX analysis of biosamples when electron imaging only is not enough!

Read the full article : https://www.nature.com/articles/srep45970

Additional examples:

https://www.nature.com/articles/s41467-020-16287-5

https://onlinelibrary.wiley.com/doi/epdf/10.1002/ejhf.1154

What are some advantages of this technique that make it suited to addressing this type of question?

Ben: The advantages of this technique are that we can elucidate the location of several critical elements within the sample, which really facilitates interpretation of ultrastructure. So, if you are working with nano-scale structures, I would recommend trying it. Of course, sample preparation is key to getting the best results. In this Pirozzi et al., 2021 article, we compiled some pointers on sample preparation: https://www.sciencedirect.com/science/article/abs/pii/S0091679X20302065

What other services do you provide in your facility that would be useful in combination with this type of imaging?

At the Correlative Light Microscopy Dutch Flagship Node we offer the following services which could all be useful for your EDX project:

• Large-scale EM (’nanotomy’) nanotomy.org

• Advanced light microscopy (light sheet, CLSM, MPE, Raman etc) umic.info

• Correlated microscopy (CLEM) umic.info

  
  

So, please get in touch!

How to apply:

EDX is part of the Euro-BioImaging Proof-of-Concept study - and it's available at the Correlative Light Microscopy Dutch Flagship Node and other Nodes. The Proof-of-Concept study makes it possible to introduce exciting, new imaging technologies to our portfolio that were previously unavailable via our network. We are currently accepting applications to use these technologies at participating Nodes as part of the Proof-of-Concept study. Be part of this study - and contribute to community-wide continuous technological innovation!

All scientists, regardless of their affiliation, area of expertise or field of activity can benefit from Euro-BioImaging’s pan-European open access services. Potential users of these new technologies are encouraged to submit project proposals via our website. To do so, you can Login to access our application platform, choose the technology you want to use and the facility you wish to visit, then submit your proposal. All applications will be processed by the Euro-BioImaging Hub. As usual, users will benefit from advice and guidance by technical experts working at the Nodes, training opportunities, and data management services.

For more information: info@eurobioimaging.eu