The New (UA)ge - Alternatives to the use of Uranyl Acetate in Electron Microscopy

Session

FIB Applications & EM Sample Prep Techniques in Biological Sciences

Authors

Beatriz Tomaz (2), Sofia Pacheco (1), Ana Laura Sousa (2), Erin M. Tranfield (2)

Affiliations

1. Instituto de Investigação e Inovação em Saúde
2. Instituto Gulbenkian de Ciência

Keywords

Uranyl Acetate, UAR-EMS, UA-Zero, Neodymium, Alternative, Electron Microscopy, Negative Staining, Conventional Processing, Post-Staining.

Abstract text

The present study aims to overcome the dilemma surrounding the use of Uranyl Acetate, by testing and comparing different reagents to find a fitting alternative. 

Uranyl Acetate is one of the most frequently used reagents in Transmission Electron Microscopy. Known for its versatility, the applications range from staining of thin sections in plastic-embedded tissues or cell pellets, to high-contrast negative staining of biological material (1).  Its salts combine with the phosphate groups in nucleic acids as well as the phosphate and carboxyl groups on the cell surface – providing electron density to the sample and therefore contrasting the various cellular elements (2,3).  

Despite most radioactive nuclides being depleted from uranyl salts used in Electron Microscopy, the chemical is still classified as radioactive. It is highly toxic, particularly in powder form, and subsequently needs a lot of precautions surrounding it’s use (2). These features necessitate the purchase of limited amounts of the reagent after a tedious bureaucratic process, followed by a notorious difficulty for the storage and disposal (1). 

The establishment of a non-hazardous alternative in the Electron Microscopy community, would decrease the health and environmental issues and bring benefits, not only to those who work with Uranyl Acetate but also for the surrounding population and biome.  

With this purpose in mind, three non-toxic reagents – UAR-EMS, UA-Zero and Neodymium – were tested alongside Uranyl Acetate, to evaluate their performance in Negative Staining Conventional Processing and Post-Staining.  UAR and UA-Zero were selected because of their easy availability on the commercial market and the reported good results with these reagents (1,4).  Neodymium was selected after excellent reports in the EM community of its suitability to replace Uranyl Acetate (5).  The trials were preformed comparing fresh reagents and 2-week-old reagents side by side; as well as following the instructions and recommendations in literature for each reagent. For the Negative Staining tests, we selected E. coli, Lactobacillus spp. and HIV-VLP samples, while for the Conventional Processing and Post-Staining the tests used 793T cell pellets. The Conventional Processing and Post-Staining will later be repeated for tissue samples. 

After the image collection in the Tecnai G2 Spirit BioTWIN Transmission Electron Microscope, using the Olympus-SIS Veleta CCD Camera, an evaluation key was created and distributed amongst various blind observers in and out of the field of Electron Microscopy.  

Once we have the results from the blinded evaluations, the mean of each score attributed will be calculated for each reagent and condition tested. We will perform a statistical analysis on the mean.

Preliminary results point to UAR-EMS being a strong contender, followed by Neodymium and with UA-Zero showcasing the weakest performance out of the four. The current hypothesis points to the lack of a “one size fits all” answer - for some samples and some protocols there seems to be a better performance of one alternative reagent over Uranyl Acetate, while in other experiments Uranyl Acetate appears to be irreplaceable. In the latter case, our best option may be to ration the remaining Uranyl Acetate for these very specialized protocols. 

References

1 - Nakakoshi M, Nishioka H, Katayama E. New versatile staining reagents for biological transmission electron microscopy that substitute for uranyl acetate. Journal of Electron Microscopy. 2011 Dec 1;60(6):401–7.

2 - Suvarna KS, Layton C, Bancroft JD. Theory and practice of histological techniques. 7. ed. Edinburgh: Elsevier Churchill Livingston; 2013. 637 p.

3 -  Hayat MA. Principles and Techniques of Electron Microscopy. 3rd edition. Basingstoke: Palgrave Macmillan; 1989. 480 p.

4 - Pinto AL, Rai RK, Shoemark A, Hogg C, Burgoyne T. UA-Zero as a Uranyl Acetate Replacement When Diagnosing Primary Ciliary Dyskinesia by Transmission Electron Microscopy. Diagnostics. 2021; 11(6):1063. https://doi.org/10.3390/diagnostics11061063 

5 - Kuipers J, Giepmans BNG. Neodymium as an alternative contrast for uranium in electron microscopy. Histochem Cell Biol. 2020 Apr;153(4):271-277. doi: 10.1007/s00418-020-01846-0. Epub 2020 Feb 1. Erratum in: Histochem Cell Biol. 2020 Dec;154(6):683.