In 2014, two Americans, Eric Betzig and William E. Moerner, and Stefan W. Hell from Germany were awarded the Nobel Prize for development of a fluorescence microscope of very high nanoscopic resolution (called super resolution) and for advancement of this research technology.

– Optical nanoscopy revolutionised the concept “seeing means believing” but limitations of field of vision and the necessity of using fluorescent labels have shown that there is still room for improvement – says Professor Maciej Trusiak.

Be gentle with cells

Cells are transparent. It is necessary to label them with fluorophores, which simply “shine” under the microscope like nanoscopic light bulbs. They absorb energy and then give it back as radiation. – For cells, especially live ones, fluorescent colouring is a source of stress. In addition, it is costly and time-consuming – stresses Professor Maciej Trusiak. – Scientists are trying to image them equally well, with similarly high contrast but without the need to label them. My team and I are participating in such research – he says.

Wide field of vision

In biological and biomedical studies it is vital to image live cells without intervention but also in a wide field of vision. A standard microscope provides high resolution from one to five cells in the whole culture. One must be lucky to be at the right time and place in which cells behave in a way interesting for biologists and other scientists, biomedics and laboratory technicians.

 – In our research we use phase microscopy started by a Physics 1953 Nobel Prize laureate, Frits Zernike. We are sensitive to not how a sample absorbs light or how it emits it but how it refracts light. In this case we can observe cells with high contrast without any labelling – explains the scientist. – Another thing is that we do not use microscopic lens since they entail limitations of resolution, field of vision and depth of focus – he adds.

NaNoLens

A method which will naturally solve the problem of limited field of vision is lensless holographic microscopy. It uses the physical foundations of in-line holography, introduced by Dennis Gabor, who was awarded the Nobel Prize in Physics in 1971 for it. A computer reconstruction of a hologram is conducted in a full-sized sensor to obtain information about the object without using labels.

The whole system consists of a camera that records the image (hologram); in front of it there is the studied sample, which is illuminated by a light source. Part of the light is dispersed on the cell, providing information and part of it remains not dispersed. Two beams make the refocused hologram, which is recorded on camera. We do not see the sample, however, but its holographic shadow.

– We can numerically imitate propagation of light in space. After hologram reconstruction (in silico propagation) we see an image similar to this from a standard microscope as if we were physically adjusting the focus but in this case we do it on the computer – explains Professor Maciej Trusiak. – We numerically reconstruct previously recorded holographic data. With an algorithm, calculations, we get from them what cannot be seen, so the quantitative phase contrast of a sharp sample – he adds.

Professor Maciej Trusiak with Piotr Zdańkowski, PhD, Julianna Winnik, PhD and doctoral students: Mikołaj Rogalski, MSc, Piotr Arcab, MSc in the laboratory

Ultraviolet will break barriers

The main challenge to overcome in lensless holographic microscopy is low spatial (ca. 1 micrometre) and in-line resolution (ca. 3 micrometres).

– Resolution tells us how close two elements may be for us to be able to see them separately and not blurred into one object. The higher the resolution, the more information we can have about a given structure and image it better – says Professor Maciej Trusiak. – In the NaNoLens project, I want to implement lensless holographic nanoscopy in deep UV as a compact solution which may be easily applied inside an incubation chamber and outside the laboratory – he mentions.  – I am going to use a shorter wavelength, ultraviolet, because it directly correlates with resolution and scattering. The shorter the wavelength, the higher the resolution – he adds.

In standard microscopes, the construction of the device and the path travelled by the beam cause the ultraviolet to be absorbed so the source of light disappears. Moreover, ultraviolet in doses that allow classical imaging is harmful to cell DNA, which can lead to their death. How is the scientist planning to prevent this?

– First, when we work with a lensless holographic microscope, we do not use any optical elements so nothing absorbs ultraviolet except for the sample – answers Professor Maciej Trusiak.  – As for the dose, we have already learnt that we need to record not so many photons in the camera field for numerical reconstruction to be successful. We will be testing how low this light might be and whether we can find such that will not harm cells at all – he says.

12 thousand microscopes in one

The scientists want to expand current knowledge on extracellular vesicles dynamics (nano-bio objects emitted by all cells) within live cell cultures, with unique precision at the level of single vesicles. The project will be carried out in the Quantitative Computational Imaging (QCI LAB) Group led by Professor Maciej Trusiak comprising: Professor Krzysztof Patorski, Professor Michał Jóźwik, Piotr Zdańkowski, PhD, Julianna Winnik, PhD and doctoral students: Maria Cywińska, MSc, Mikołaj Rogalski, MSc, Piotr Arcab, MSc and Emilia Wdowiak, MSc.

Professor Maciej Trusiak with part of the Quantitative Computational Imaging QCI LAB research team

The team of Professor Trusiak conducts extensive international cooperation with scientists from the University of Valencia (Spain), the University of Nanjing (China) and the Arctic University of Norway in Tromsø and domestic cooperation with researchers from institutes of the Polish Academy of Sciences: Nencki Institute of Experimental Biology and Mirosław Mossakowski Institute of Experimental and Clinical Medicine. The cooperation will be continued.

– If everything goes well, it will be possible to image in such a wide field that would otherwise be obtained by connecting fields of over 12 thousand phase microscopes currently available on the market – stresses Professor Maciej Trusiak. – As for information capacity, now calculated in megapixels, I will want to achieve the level of tens of gigapixels – he adds.

To complete the project ”Lensless label-free nanoscopy” (NaNoLens) the scientist was awarded 1.5 million Euro within the ERC Starting Grant, given by the European Research Council (ERC). The research will start in January 2024 and will last 5 years.

Please turn on English subtitles in the video.