Quantitative Mapping of Mercury and Selenium in Mushroom Fruit Bodies with Laser Ablation–Inductively Coupled Plasma-Mass Spectrometry

Some macrofungal species (mushrooms) are known for their ability to accumulate high concentrations of mercury in their fruit bodies, especially Boletus edulis and its close relatives Boletus pinophilusBoletus aereus, and Boletus reticulatus. This is of special interest, because these species (more commonly known as porcini mushrooms) are some of the most sought and consumed types of wild-grown mushrooms worldwide. A recent paper by Simone Braeuer and colleagues of the University of Graz (Austria) and Ghent University (Belgium) discusses an efficient method developed for quantitative mapping of mercury and selenium in mushroom fruit bodies via laser ablation coupled to inductively coupled plasma–mass spectrometry (LA-ICP-MS) with excellent limits of detection (LODs) and high spatial resolution (down to 5 µm). Braeuer spoke to Spectroscopy about this paper.

Your paper (1) deals with the development of a novel method for quantitative mapping of mercury and selenium in mushroom fruit body tissues using LA-ICP-MS. What benefits has this method created for researchers in general, and for ICP-MS spectroscopists in particular?

Generally speaking, our developments enable the investigation of the distribution of heavy metals and trace elements in biological tissues, with a high spatial resolution, nice limits of detection, minimal sample preparation, and complete analysis in a very short amount of time. In addition, actual concentrations can be obtained, not just mere qualitative information in the form of crude intensities. Thus, our method will be highly beneficial for the investigation of the pathways of various elements in nature. Further, our method is the first that particularly addresses the poor stability and the “stickiness” of mercury during analysis using LA-ICP-MS, and we found surprisingly simple but effective ways to minimize these problems, which also work very nicely for selenium.

Why were mercury and selenium selected as constituents for your measurements in mushrooms?

We originally selected these two elements because porcini mushrooms are accumulating them in high concentrations in nature. This is quite fascinating, because selenium is often regarded as natural antagonist of mercury, mitigating its toxic effects. Terrestrial organisms accumulating both elements at the same time are rather rare. What makes it even more worth researching is the popularity of porcini mushrooms: They are amongst the best known and most appreciated edible mushrooms, enjoyed by many mushroom lovers.

Further, mercury and also selenium were interesting for us from an analytical point of view, because they turned out to behave very differently than most other elements in an LA-ICP-MS setup; both have a much slower washout time (or “single pulse response”), which means that specific care has to be taken if high quality results are to be obtained.

Briefly describe your discovery and development process, and how this differed from what was previously done by yourself or others.

I have investigated mushrooms before, but never with LA-ICP-MS. Also in the whole research community, most work concerning elements in mushrooms is focusing on the determination of the bulk concentrations. It is known that porcini mushrooms can contain high levels of mercury and selenium, but nobody knows why and how the two elements are taken up and then distributed in the fruit-bodies. Since LA-ICP-MS has undergone significant improvements in the last decade or so, we decided to check if we could use the technique to get more meaningful information about elements in mushrooms, more specifically their spatial distribution. I believe that our work profited tremendously from the close collaboration between chemists and biologists. Analytical chemistry is awesome, but only the expertise of biologists and the constant exchange with each other allowed us to obtain such excellent results. It is a great example of what can be achieved in environmental research through interdisciplinarity collaboration.

Please summarize your findings.

Actually, before we could work on the content of the paper you mentioned in the beginning, we focused on the optimization of the instrumental setup, for example the LA parameters. In the end, we mainly adapted small, but significant things. For example, the length and inner diameter of the tubing connecting the LA and the ICP-MS unit has a significant influence on the washout behavior of mercury and selenium. We published a systematic investigation of these settings as a short technical note (2). We were able to optimize the instrumental setup and settings, so that the washout time of both elements was significantly shortened, which means that the measurements are much faster. This allowed us to obtain mercury and selenium maps of several different mushroom samples, and of different parts of each fruit-body, in a comparatively short time (1). To prepare the samples, we cooperated with biologists, who were able to prepare high-quality 10 µm thin sections for us, without the need for chemical fixation, which would have likely significantly affected the element distribution in our samples.

We originally used a laser spot size of 20 µm, which already resulted in useful maps, where we could see that mercury, and also selenium, were much more concentrated in the fruit-bodies’ peripheral tissues (or the “skin,” if you like) than in the adjacent inner context tissue. In some cases, this higher-concentrated layer was only around 40 µm thick, and the border to the context tissue was unexpectedly sharp. This has never been observed before, and we have no explanation for it at the moment. We were also able to further reduce the laser spot size down to 5 µm, and obtained even more detailed mercury and selenium maps of our samples, indicating the high potential of our method for future in-depth investigations. However, since a decrease of the laser spot size goes hand in hand with a poorer limit of detection, this small spot size was only possible because porcini mushrooms contain such high amounts of mercury and selenium. But don’t worry, we calculated that you could eat roughly 0.5 kg of porcini mushrooms every week, all year around, and would still not exceed the recommended maximum doses of mercury and selenium.

Another significant part of our paper deals with the development of a proper quantification strategy. We prepared small droplets of a gelatin matrix that contained defined amounts of our analytes (mercury and selenium), which is one of the most common approaches for quantification with LA-ICP-MS. However, we observed significant instabilities and inhomogeneities, which would have rendered quantification impossible. Finally, after some initial unsuccessful approaches, we just added some L-cysteine, which is known to work as a complexing agent for mercury. And indeed, it led to tremendous improvements for the quantification of mercury, and also selenium. So easy! Finally, for proper quality assurance, we prepared an in-house reference material from a mushroom powder, for which we got excellent results.

What are some of the benefits in using LA-ICP-MS in mapping mercury and selenium as opposed to other spectroscopic methods?

With LA coupled to ICP-MS, it is possible to get spatially resolved information of almost all elements of the periodic table. For some research fields, this can be really beneficial, for example when investigating the biogeochemical cycle of trace elements. We can find out where exactly the individual elements are stored, concentrated or excluded, or even follow transport routes. Compared to other mapping or imaging techniques, LA-ICP-MS requires only very little sample preparation, and the limits of detection are usually much better, depending on the element, and spatial resolution. One of the Achilles’ heels of LA-ICP-MS has always been the quantification and quality control, but there have been nice improvements lately, and the method we developed works excellently, with rather little effort.

Were there any particular challenges you encountered in your work?

Well, the Covid-19 pandemic hit right when we wanted to start with the lab work for the project. Due to the strict regulations of the university, especially in the beginning of the pandemic, it took more than half a year until we could finally start for real. Also, we expected that mercury would be problematic because its stickiness is well known, almost infamous, among analytical chemists. However, we were really surprised when actually selenium turned out to be almost as difficult to handle as mercury in our LA-ICP-MS setup. Especially quantification was challenging in the beginning, due to its inhomogeneous distribution in our calibration standards.

What sort of feedback did you receive regarding this work?

The paper has only been published very recently, but, so far, people really appreciate our work. They especially like our conclusion that porcini mushrooms can be consumed (in “normal” amounts) without having to worry about mercury or selenium intoxication. This actually also includes myself, I was really happy when our calculations showed that I can continue to enjoy a nice dish of porcini mushrooms once in a while.

Do you believe that this method can easily translate to other food types and to other elements, and, if so, which testing of food types do you believe would result in the most beneficial translations?

Yes, this should be possible without difficulties. It would be very interesting to apply the method to plant roots, and to mycorrhizae–the interface between fungus and plant. It could also be worthwhile to use our method for the investigation of food with problematic concentrations of toxic elements, for example to discover the specific storage locations. This would facilitate the search for methods to decrease the element’s content and make our food safer for us consumers.

What are the next steps in this research?

At the moment, we are looking into decreasing the spot size as much as possible, to get an even better spatial resolution. Ideally, we would like to be able to distinguish individual fungal cells from each other. This would allow a much better insight into the element transport and storage strategies of the mushrooms. A better spatial resolution would also mean that we could look for a co-localization of mercury and selenium, to verify the antagonistic role of selenium.

Further, we also want to adapt our method, to investigate the distribution of other elements in mushroom fruit-bodies. Until now, hardly anything has been researched in this field, and there is a large gap of knowledge about the translocation and storage of metals and trace elements in naturally grown mushrooms, although fungi are important constituents of our environment. They fulfill many different important tasks in the ecosystem, and thus absolutely deserve more attention from all kinds of research areas, including atomic spectrometry.

References

(1) S. Braeuer, T. Van Helden, T. Van Acker, O. Leroux, D. Van Der Straeten, A. Verbeken, J. Borovička, and F. Vanhaecke, Anal. Bioanal. Chem. 1–14 (2022). https://doi.org/10.1007/s00216-022-04240-y

(2) T. Van Helden, S. Braeuer, T. Van Acker, O. Leroux, D. Van Der Straeten and F. Vanhaecke, J. Anal. At. Spectrom. 1455–1461 (2022). https://doi.org/10.1039/D2JA00131D

Simone Braeuer received her PhD in 2018 in analytical chemistry from the University of Graz, Austria, under the supervision of Professor Walter Goessler. During her dissertation, she worked on the investigation of arsenic and its various compounds in macrofungi, using ICP-MS and HPLC coupled to ICP-MS. In 2019, she obtained the “Erwin Schrödinger Fellowship” from the Austrian Science Fund, FWF, and joined the Atomic & Mass Spectrometry group of Professor Frank Vanhaecke at Ghent University, Belgium. There, she used and optimized several different ICP-MS based methods to explore different aspects of mercury and selenium in biological samples, mostly mushrooms, including mercury isotope ratios, speciation analysis and the spatial distribution of the elements in fruit-bodies. She returned to the University of Graz in May 2022, where she is currently working as a senior scientist. Her research focuses on the development and further application of analytical methods to investigate the biogeochemical cycle of trace elements, especially in and around macrofungi.