21. Feb. 2024

Associate Professor Pavel Pořízka is a member of Prof. Kaiser's research group, Advanced Instrumentation and Methods for Materials Characterization, at CEITEC BUT. Although the researchers mostly focus on the development of instrumentation and methodology in collaboration with industry, they have recently started to explore applications in biology thanks to a junior grant. In a GAČR-funded project entitled Processes of laser ablation of soft tissues and subsequent development of laser-excited plasmas, Pořízka and his team focused on the analysis of soft tissues using LIBS (Laser-Induced Breakdown Spectroscopy).

The completed project was rated as excellent; it was also the first grant you led. Can you tell us more about it?

That was under a programme that I am happy to talk about and of which I was a successful applicant. It's a good thing that grant agencies provide individual calls for young scientists at the beginning of their careers, give them the opportunity to participate in a grant competition and gain valuable experience in managing larger projects. The grant we are talking about was announced by the Czech Science Foundation (Grantová agentura České republiky, GAČR) in the GAČR JUNIOR call (which gradually transformed into the larger JUNIOR STAR call). It's basically financial support for budding scientists. One of the conditions for applying for this grant was a long-term internship abroad, so I went to the University of Florida under a Fulbright grant.

Thanks to the junior grant, I had the opportunity to build my own team focusing only on the analysis of biological samples and to be its principal investigator. Until then, I was mainly a co-investigator on grants within our research group. As was the case of the previous grant awarded to my colleague Pavlina Modlitbová, which was again a grant for young researchers, TAČR – ZÉTA programme. This grant focused mainly on applied research, but it was in the field of biology, too.

What do you see as the biggest advantage of these programmes that support scientists at the beginning of their careers?

They literally get careers off the ground. Usually, if you haven't had a grant and you haven't been a principal investigator, you have little chance of getting a grant, which creates a kind of vicious circle. These programmes are supposed to disrupt that established system, and I'm glad it's working that way. If there is even more intensive funding for grants for early-career scientists and, in particular, female scientists, that will only be a good thing. Thanks to this programme, we have received follow-up grants that have helped us to enter other scientific fields. It really is a great helping hand at the beginning of a career.

How does the process of selecting winning projects work?

Practically, it's a standardized procedure. The project is evaluated by a GAČR panel, where the committee and the opponents give their opinions on it. It is not simply included in the regular GAČR cycle with standard projects; it is judged outside of it. As a result, the project is evaluated independently of other project applications submitted by experienced scientists with a "better" CV. The h-index has long been crucial from the GAČR's point of view but this is also changing. At the beginning of one's career, one has a less impressive CV, and when one has to compete with someone who has a successful 20-year-long scientific career and has already received several research grants in the past, it is hard to get ahead.

Why did you take an interest in biological applications?

After returning from my internship abroad as part of my PhD studies at BUT, in our research group, we started to develop not only the development of instrumentation, which was historically strong here, but also the possible applications. This direction was interesting for several reasons. The most important factor was that it was a topic that I was personally interested in, especially in terms of the difficulty of analysing the sample itself, interpreting the data, and optimizing the parameters of the measurement setup. It was also something that pushed our collaboration towards developmental biology and, in time, perhaps clinical research. Thanks to this, we are collaborating with the Academy of Sciences and clinical departments at the University Hospitals in Hradec Králové and Ostrava. Last but not least, we have moved on to another grant and it has also brought us new contacts abroad.

Can you highlight any foreign cooperation?

The strongest foreign cooperation in the field of biology is with the research group of Professor Andreas Limbeck from the Technical University of Vienna and Associate Professor Gabriela Kalčíková from the University of Ljubljana. We have a joint grant with both on the detection of microplastics in soft tissues and their influence and toxicity in these tissues. To sum it all up, this was a strategic and key decision for us – we have expanded our existing applications to include biology, and this has taken us further. We've created a smaller team of about five or six students and two postdocs that are just focused on biological applications, whether we're talking about plant analysis, hard tissue (teeth and bones), or soft tissue (organs).

What is the potential of LIBS in the study of tumour tissues?

LIBS is a method of analytical chemistry designed for elemental analysis that is applied in a wide range of fields, including biology, but also, for example, geology. The method allows the distribution of elements on the surface of a prepared sample to be measured. In clinical research, we are investigating whether the LIBS method can be used to diagnose and support pathology and standard procedures currently used to refine the estimation of the extent of cancer in human skin. This is our main hypothesis. If we are able to refine the estimation of the area of diseased tissue, this could be used for diagnostic purposes during surgical procedures in the future. However, this remains a vision for now.

How far are you?

At the moment, we have already obtained dozens of samples from the hospital, and we have permission from the ethics committee to study them. The analysis of these samples is ongoing and we are preparing several scientific articles to present our research achievements. We also participate in international conferences where we receive positive feedback. This support convinces us that our work is worthwhile and that we should continue. We are focusing on combining different approaches from the perspective of correlative microscopy, which means combining classical microscopy and spectroscopy to use different techniques to obtain the most comprehensive information about the samples, i.e. how the chemical and structural composition changes depending on the disease being studied.

Where would you like to take the research in the near future?

We are moving forward and would like to submit another grant in the framework of Czech-Austrian cooperation in collaboration with the Technical University of Vienna. This will be a follow-up grant focusing on human tissue analysis and the study of skin cancer. But again, I have to stress that the beginning of this journey was the junior grant mentioned above which opened the door for us both in soft tissue analysis and international cooperation. Without this support, we would not have been able to successfully continue to discover new applications and expand the use of our method.

So you have analysed mouse and pig tissues first?

Clinical samples are very valuable to us, so we have model tissues – organs from laboratory mice – that we use to simulate human tissue. We don't measure clinical samples straight away, we first have to check if the system itself is working at maximum performance. In fact, when we analyse samples with LIBS, we use a powerful laser pulse to slightly heat and vaporise the material, creating the necessary analytical glowing plasma, thus practically destroying the sample. So the thin histological sections we get from the hospital are gone after the measurements.

Can we say that you are pioneers in measuring tumour tissue with LIBS?

I wouldn't say that at all. There are a few labs in the global community working on similar topics. We are slightly inspired by the success of our French colleagues at the University of Lyon. The LIBS method is not as established as competing methods. This is what makes the whole research more interesting, as we have to discover a lot of things ourselves. We are trying to move it closer to standard, routine use. So I wouldn't say pioneers, but I think we are making a significant contribution to making it more widely adopted and more competitive with other techniques.

Does the startup you founded in 2019, Lightigo, help you do that?

Extremely. It helps us develop systems that are not commercially available in the world, which was one of the most significant limitations of the whole method. This is something where we see ourselves quite at the forefront – on the one hand, we are developing the actual method of how to measure samples, and on the other hand, we are also able to develop systems in broad collaboration with our startup. This connection gives us the unique ability to adapt the system according to how it should measure, what performance it should have, which also makes it possible for us to move forward faster. This way, we are not dependent on any third-party instrumentation. However, it's more challenging in that we need broader funding.

How demanding, financially speaking, is LIBS compared to competing methods?

The instrumentation and the low cost of the method are some of its biggest advantages. Our system is significantly cheaper than competing techniques, such as LA-ICP-MS, but lacks the sensitivity that seems to be essential in some applications. It is sufficient In applications such as the detection of heavy metals in teeth and bone. Although the LIBS method cannot be adopted in all applications, it can be used in many cases and achieve both financial and time savings. The limitations of the method were one of the aspects we addressed in the JUNIOR grant.

Are there any applications where you have already got into “hot water”?

There are some but that makes it all the more interesting to try. For example, we have not yet been able to detect iodine, which is used to stain the sample before computed tomography, i.e. before structural analysis. But we don't see it as a failure. It shouldn't sound like that, because if we can't do something now, it doesn't mean that we won’t be able to do it in the future with further development of instrumentation and measurement methodology.

What is the priority project now?

Right now, the three most important projects for us are the three GAČR projects and other TAČR projects in the TREND and NCK programmes. So we have a number of ongoing projects that involve the development of instrumentation and application in industry. However, we are able to manage such a broad scope because we have a group of talented students in our lab who help us significantly in moving forward quickly. They work on sub-topics and actively contribute to the development of our method during their studies.

Two GAČR projects are focused directly on biology, meaning we are able to follow our strategy. In the first project, we are starting our third year of work, and it concerns the detection of nanoparticles as indirect biomarkers of human tissue. With this method, we can specifically label the defined tissue, for example, breast cancer. Another priority for us is the detection of microplastics in soft tissues, where we combine LIBS, LA-ICP-MS and Raman spectroscopy for chemical analysis. We complement these spectroscopic and spectrometric methods with structural analysis by computed tomography from the laboratory of Assoc. Tomáš Zikmund, who is also part of Prof. Kaiser's research group. Our aim is to find out how the presence of microplastics affects the surrounding soft tissue and whether their presence can have any negative (toxic) effects. Trace elements are commonly found in plastics, including some toxic ones such as lead and cadmium. These substances can diffuse into the surrounding tissue and possibly affect it.

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