3 November 2021
The word “super-resolution” brings crystal clear, high resolution TV wonders to mind for most of us; however, in microscopy, the term has a different meaning. It means a technology that circumvents the problem caused by the wavelength of light, making the observation of cell- or even molecular level life processes possible. We have visited the University of Pécs Medical School’s Physiology Department Nano-Bio-Imaging Centre, where – among others – research is underway into the yet unknown forms of communication between cells and its significance with Nobel-worthy technology.
written by Miklós Stemler
The Nano-Bio-Imagine Centre of the Pécs Medical School’s Physiology Department does not look particularly exciting at first glance – however, the locking doors are a sign that this is not just any place. The dimly lit room houses odd devices, flanked by workstations equipped with large screens showing images that almost look like colourful, abstract paintings. I need a few minutes to realise that the incredibly detailed images show cells; and I notice that my mild dust allergy has subsided.
Ferenc Wilhelm, the technical supervisor of the centre tells me that my lack of symptoms is due to the locking doors and the internal vent system providing positive pressure in the room – therefore, the air of the room is free of any dust. The devices in this room are highly sensitive, any dust or change in temperature can cause their settings to change, which can make taking measurements impossible. However, in order to understand what these devices are and why they can cost hundreds of millions of forints I have to take part in a short microscopy course held by one of the researchers of the centre, András Lukács, associate professor in the Department of Biophysics.
“The wavelength of light is an impenetrable obstacle for light microscopy, since it determines the maximal value of resolution, or how long we are able to differentiate between two separate points. This so-called diffraction limit can be overstepped with electron microscopes using different technology. However, this technique is limited in its use in physiology, since samples have to be frozen and under vacuum; this means that there is no chance of studying their life processes. All of this kept us from seeing the events within cells for a long time – this problem was solved by the so-called super-resolution microscopy or nanoscopy; it circumvents the obstacles of physics with tricky solutions” – András Lukács added, throwing me in the deep end.
The Nobel-worthy discovery of 2014 resulted in the appearance of various super-resolution technologies. One of these is the STED microscope: it uses to laser beams to differentiate between two points. One of them stimulates molecules that will emit light due to fluorescence, the other puts them back on their base status. The result is impressive: one side of the screen shows a blotchy image made with traditional technologies while the other side shows the structure of the pattern nicely – I am told that I’m looking at the surface structure of a cell.
Two researchers of the Department of Physiology, Dávid Ernszt and Soma Godó are using this device to study the cell membrane. While the area would look homogenous with a traditional light microscope, super-resolution opens a whole new world of various shapes of objects and structures. The researchers explain that with the help of the new microscopy technologies, we know that the cell membrane is not a homogenous surface, but its regions play an important role in signal transmission; they are studying the basic principles of this process. All of this can play a huge role in brain research; some theories suspect that these regions and their changes might have an expansive role in the development and course of neurodegenerative diseases.
The N-STORM microscope works on a different principle, stimulating molecules with randomly generated, low energy laser impulses, combining them for creating images. Next to it, the structured lighting of the SIM microscope shows images of odd threads, or the transmission nanotubes between cells. The novelty of the research and discovery is shown by the fact that the tubes re-defining our current knowledge about communication between cells have not yet been added to the medical curriculum.
My guides explain that every super-resolution solution has its pros and cons. STED allows higher resolution than SIM, but the latter can handle samples prepared in the traditional way that has a big practical importance; the N-STORM crates perfect quality images, but the randomised imagine means that creating even one picture is time-consuming and so on. The main forte of the microscopy centre in Pécs is the combination of different technologies and in their varied uses.
The nanometre domain
The resolution limit of traditional light microscopes is around a micrometre (one thousandth of a millimetre), while super-resolution can take us to the domain of nanometres (one thousandth of a micrometre). With this resolution, we can see inside the cells and the processes in-between, which has led to the discovery of the nanotubes between cells – the implications of which are yet to be seen.
The width of many of these nanotubes does not even reach 200 nanometres, therefore scientists had no idea about their existence for a long time, since they are invisible for traditional light microscopes, and these connections between living cells are not observable in samples prepared for electron microscopes, explains Edina Szabó-Meleg, researcher of the topic.
The existence of nanotubes has rewritten what we know about the processes between cells. It is also incredibly important relating to the cellular level of disease spread, since pathogens could infect cells through these tubes by circumventing the immune system – in a Pécs research cooperation with a local biotechnology company is studying exactly the spread of COVID-19 between cells as part of a tender. Providing a path for viruses is of course not the main function of the tubes; individual cells transmit mitochondria, or energy-producing cell particles to each other through them.
As most important discoveries, this is naturally raising as many new questions as it answers. Researchers in Pécs are trying to answer basic questions by studying zebrafish embryos like what kinds of cells and conditions are needed for the creation of nanotubes and what kind of materials are transmitted through them. They have already determined that “unhappy” cells (cells under stress caused by various materials and circumstances) are more likely to develop such connections, allowing cells to help each other; they might also play some role in foetal development.
A potential use case could be providing helpful substances that do not cause damage to other particles to cells “unhappy” due to a specific disease, but such practical uses are still in the far future.
Researchers in Pécs have studied the development of nanocells between cells subjected to trauma after cataract surgery. After traditional “tear-off” removal of cataracts, they have found a high number of nanotubes, while almost none after laser surgery. Another interesting titbit is that patients heal faster and with less complications after traditional surgeries causing more cellular trauma.
While new discoveries lead to more questions, monitoring the images seen under the microscopes is no child’s play either. The smallest change in the environment or misaligned setting can cause imaging mistakes that are incredibly hard to notice, since researchers are only now learning about the rules of this nanoworld – as András Lukács says, there could be entire doctoral dissertations written just about the monitoring methods. It took years for clinical doctors to even accept the existence of nanotubes between cells, since even if they were visible in the samples, they are hard to find within tissue in a living organism.
Edina Szabó-Meleg leads us further into the (seemingly) colourful world of mitochondria: they are dealing with their movement between cells right now, and this is a research direction that could be called new worldwide. Studying the movement of mitochondria between nerves could bring us closer to understand the process and hopefully the treatment of such terrifying neurodegenerative diseases as Alzheimer’s and Parkinson’s; more and more evidence shows that the degeneration of nerves is caused by the transmission of damaged mitochondria.
The pharmaceutical industry has already started the application of nanotubes; however, it is still wholly experimental: the goal is to get the active agent to the targeted cells wrapped in so-called nanobeads.
The novelty of the research area is shown by the fact that there are not many places dedicated to this research in the world. As the Pécs expert on the topic says, “I could count on my two hands the number of research places dealing with nanotubes”. Few places have the necessary equipment. Moreover, the clinical background is important for the monitoring and further development of research findings.
At the end of our journey to the nanoworld we have many questions, but one certainty: the new physiological phenomena discovered via super-resolution microscopy provide now immeasurable possibilities for the understanding of basic life functions and the treatment of yet untreatable diseases. With the words of one of my guides, super-resolution provides enough research directions for the next hundred years – meanwhile, us laymen can marvel at the beauty of living cells reminiscent of abstract paintings.
(This article was originally published on hvg.hu.)
UPMS, Lajos Kalmár