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Safer, Non-Radioactive Treatment for Viruses Undergoing Transmission Electron Microscopy

A new neutral, non-radioactive negative staining reagent for the high-resolution viewing of viruses using transmission electron microscopy has been discovered by researchers.

Ever wondered how scientists “see” biological structures at the nanoscale? To do this, scientists need to make use of transmission electron microscopy (TEM), where electrons are fired at a sample and their interactions with the sample are captured as an image.

However, during this process, samples like viruses need to be stained with a radioactive solution called uranyl acetate, which is biologically toxic. This results in a high-resolution electron microscopy image at the cost of safety and the ease of synthesis and storage of the staining solution.

Fortunately, Masahiro Sadakane, Professor of Applied Chemistry at Hiroshima University’s Graduate School of Advanced Science and Engineering, led a team of researchers to develop a non-radioactive alternative, a “Preyssler-type” phosphotungstate, to produce high-resolution images of the same quality without the safety and storage risks of uranyl acetate.

So far, other non-radioactive alternatives like the “Keggin-type” phosphotungstic acid may be used as a staining agent for biological materials for TEM. This molecule consists of a central unit of one phosphate and four oxygen atoms, which is further surrounded by tungsten and more oxygen atoms. Even though this alternative is not radioactive, it is highly acidic and must be neutralised before use. The other drawbacks of this alternative include lower resolution of the produced electron microscopy image.

Despite the drawbacks, it has been recognised that the “Keggin-type” phosphotungstic acid belongs to a large family of similar compounds – the phosphotungstates, which may be further studied to find safer alternatives that produce comparable image resolution to uranyl acetate.

The team’s research focus, the “Preyssler-type” phosphotungstates, had been reported in previous works to have feasible uses as a negative staining reagent to observe the fine structure of bacteria.

“Preyssler-type” phosphotungstates also comprise oxygen, phosphate, and tungsten, but are structurally different from “Keggin-type” phosphotungstates as the atoms surround an encapsulated positively charged ion like calcium or sodium. This results in a molecule that is more stable than the “Keggin-type” phosphotungstates, produced as a potassium salt.

The team stained three types of bacterial viruses (phages), which infect bacteria with Preyssler-type phosphotungstates, and obtained their images through TEM.

“Our results indicate that Preyssler-type phosphotungstates are good negative-staining reagents for virus observations,” Sadakane said. “They are easy to use, since they are not radioactive and do not need adjustment for pH levels, and they provide clear images.”

With their findings, the team hopes to develop other kinds of non-radioactive negative staining reagents to observe other viruses and other biomolecules such as proteins, for the ease of preparation of biological samples for TEM. [APBN]

Source: Sahiro et al. (2022). Preyssler-type phosphotungstate is a new family of negative-staining reagents for the TEM observation of viruses. Scientific reports,12(1), 1-8.