Olomouc Scientists Push the Boundaries in Plant Cells Imaging
Visualisation of cortical microtubules by means of super-resolution microscopy
Scientists at the Centre of the Haná Region for Biotechnical and Agricultural Research (CHR) and the Institute of Molecular and Translational Medicine (IMTM) have discovered how to observe living cells in unprecedented detail. Super-resolution microscopy based on the structured illumination of cells was used for the first time in history for observing living plant specimens. This allows observation of cell processes in motion – and at a resolution unthinkable in microscopy not so long ago.
The study, published in the prestigious U.S. journal Plant Physiology, is the result of an approximately three-year-long collaboration of the Department of Cell Biology at the CHR and the Laboratory of Genomic Integrity at the IMTM, led by Prof. Jiří Bártek. “Until now, scientists have only been able to use this method in microscopy on dead plant cells, whereas we are able to use living specimens, increasing the resolution to one hundred nanometres. The boundary of 200 nanometres, the Abbe diffraction limit for illumination microscopy, has until recently been considered an impenetrable physical barrier. We are in new territory here,” said Prof. Jozef Šamaj, Head of the Department of Cell Biology at the CHR.
“Videos” instead of static images
Olomouc scientists focussed on the structure, organisation, and dynamics of microtubules, the system of miniature tubes which are components making up the cytoskeleton (inner cellular skeleton). “The cortical microtubules in the cell membrane were the centre of our attention. They affect the way cellulose is stored in the cell wall, and thus how strong the cell wall, as well as the whole plant, will be. The orientation of microtubules also determines which direction the plant will grow,” explained Šamaj.
Until recently, scientists had to settle for images of dead (fixed) cells only, whereas now they are able to monitor the microtubules in motion. The specimen is recorded through a special light grid, which rotates and projects a typical pattern onto the images, called a moiré. By means of mathematical and statistical methods, the individual images are connected, and the resulting image is much more detailed and sharper than ever before. “It is really a huge step forward. We can count the microtubules on the basis of the level of fluorescence, and distinguish how close to each other they are. Nobody has been able to do this before with living cells,” emphasised Šamaj.
Huge potential for applied research
According to Šamaj, these findings provide important information concerning the development of plants and their resistance to stress. This could be practically applied in the cultivation or genetic modification of major crops in the future. “It is a very remote goal, but we do have some information now. We need to understand first how the cell works, and then we can proceed to applications. The cell is the smallest factory in the plant,” said Šamaj, suggesting new directions of the research. The plant cells serve, in this particular case, as an ideal model, but this method could also be used in studying the cell dynamics in medical research, which is the domain of the IMTM.
The researchers worked with the model plant Arabidopsis for a number of months. The first stage involved the preparation of the specimens themselves. “We had to test in what conditions the plants behave naturally so that we could analyse them microscopically. This took us one year,” said one of the co-authors of the study, George Komis from the CHR, describing the beginnings of their research. Microscopic recording took another year, due to the high demands concerning the quality of the specimens and stability of the external environment. This was made possible mainly by the microscopic experience of Martin Mistrík from the IMTM. Subsequent evaluation was quite arduous and time-consuming as well. The scientists had at their disposition a device allowing four methods of imaging, which they compared. In the years to come, they intend to move on from Arabidopsis to barley or alfalfa in their exploration of microtubules.
