One of the most important tools in cell biology research is what is known as a fluorescence microscope. With the aid of such a microscope it is possible to identify exactly the location of specific proteins in the cell, how they move and whether they interact with other proteins.
However, illumination from the microscope can cause cellular damage and, in doing so, alter the measurement results. Katarina Logg, who recently completed a PhD at Chalmers, has investigated this phenomenon in yeast cells and can now provide advice on how to minimise the risk of illumination-induced cellular damage.
“My results show that the cells are not affected by illumination doses below 0.16 joules per square centimetre , applied every 30 seconds for one hour. If it is necessary to use stronger illumination than this, it is best to spread the illumination dosage or prolong the exposure period, as it exerts less stress on the cells than a short exposure period.”
She produced her results by investigating a special protein which is a well-known stress indicator in yeast cells. If this protein is activated it is known that the yeast cells are exposed to stress and thus potentially behave differently than normal. The results showed that yeast cells are stressed by considerably lower illumination levels than expected from examination of the cells’ growth or appearance.
The results will be published in a scientific journal, FEMS Yeast Research, and Katarina Logg explains that they fill a knowledge gap in fluorescence microscopy.
“Very little research has been done on how illumination affects the cells and it is easy to believe that the cells are not affected if you cannot find any changes in growth and appearance. When I began my research, no instructions were available on how to avoid illumination damage when imaging cells using time lapse fluorescence microscopy.”
Her supervisor, Mikael Käll, says that it is interesting that normal budding yeast reacts to such low illumination levels.
“Budding yeast is one of the most common model organisms in cell biology. We now know that these cells are affected by a surprisingly low illumination intensity, considerably lower than what we had expected. It is quite possible that other cells, such as human cells, demonstrate a similar stress reaction to illumination from a microscope.”
Katarina Logg is his first PhD student in an interdisciplinary collaborative venture where his research group in bionanophotonics is co-operating with biologists and mathematicians.
The thesis also presents algorithms for automated image analysis in conjunction with optical microscopy. These algorithms are included in a new software package for image analysis, developed through co-operation between the Fraunhofer-Chalmers Research Centre for industrial mathematics and Mikael Käll’s research group.
The software package can be used to carry out quantitative investigations using optical microscopy. At present, image analysis is normally done manually, although manual analyses are often qualitative and subjective. With a quantitative analysis method, more objective results are obtained and more resource-intensive analyses can be made, such as measuring the spread of a certain protein in a whole cell population over time.
The thesis “Development of quantitative optical microscopy methods for single cell analysis” was defended on April 3.