Periodic Reporting for period 4 - Stress Imaging (Nanoscale Stress Imaging with Imperfect Diamonds)
Période du rapport: 2021-09-01 au 2023-08-31
Free radicals play a role in several processes in healthy individuals including how we defend ourselfes against bacteria or viruses, how cells talk to one another or how our cells age. But they are also present, when a cell responds to stress for instance when a cancer cell is killed by a drug. However, depite their importance it is difficult for the state of the art to measure free radical generation since they are short lived, reactive and low in abundance. In the ERC project we used quantum sensing to measure these free radicals in living cells. The new technology allows us to measure in which organelles free radicals are built during a stress response. By measuring how these special diamonds change their brightness we can extract information that is similar to an MRI. With the difference, that we can see much smaller signals. Using this method we were able to differentiate between young and old cells. This was demonstrated in yeast cells which function as an ageing model. Since our method is based on nanodiamond particles which need to enter the cells, we first optimized particle uptake into yeast cells. While this is trivial for mammalian cells which readily ingest particles, uptake is challenging in yeast since they have a thick cell wall.
We were able to quantify and compare the free radical load in young and old cells. We also investigated different mutant strains which are different in their longevity and found clear differences. Interestingly, mutant cells do not necessarily have a lower radical load but respond to stress differently than wild type cells. Further, we were able to age cells under different conditions which is interesting for ageing research.
We were also able to measure how much of these radicals our immune cells generate when we are stimulating them. More specifically, we were able to target nanodiamonds to specific locations as for instance the mitochondria and measure specifically at these specific organelles.
The versatile technique has since been used for measurements in patient samples, tissues and body fluid. Recently, the startup company QTsense has been founded to exploit the knowledge generated in this project.
We were able to quantify and compare the free radical load in young and old cells. We also investigated different mutant strains which are different in their longevity and found clear differences. Interestingly, mutant cells do not necessarily have a lower radical load but respond to stress differently than wild type cells. Further, we were able to age cells under different conditions which is interesting for ageing research.
We were also able to measure how much of these radicals our immune cells generate when we are stimulating them. More specifically, we were able to target nanodiamonds to specific locations as for instance the mitochondria and measure specifically at these specific organelles.
The versatile technique has since been used for measurements in patient samples, tissues and body fluid. Recently, the startup company QTsense has been founded to exploit the knowledge generated in this project.
The first part of the project was dominated by equipment building as well as optimization of samples. However, we were also already able to obtain first results on biological samples.
So far we have performed T1 measurements on yeasts and macrophages as well as reference samples. In macrophages we were able to follow nitric oxide signaling. There we are able to measure triggering and inhibition of free radical production. We also have already achieved to gain some control over the particle location within the cell by attaching antibodies to the diamond surface. So far co-localisation with the nucleus or with mitochondria has been shown.
In yeast cells we were able to improve diamond uptake and to diffententiate between young and old cells, between different knock-out strains. Additionally, we compared cells that were aged in presence or absence of an antioxidant.
The measurements on test samples helped us to identify which particles and which data analysis method are best to use . Additionally, we could learn from this data what our signals mean.
We have implemented 3D particle tracking was essential record a signal at all from a particle that is moving. However, particle trajectories give interesting additional insights. How fast a particle is moving inside the cell can for instance indicate where within a cell it is. Additionally, one can use the natural movement to obtain information on different parts of the cell. On our way to differentiating between radicals we have adapted the measuring program for pulsing and upgraded the equipment in the group.
For all the experiments that we have done with our technique we have also compared with the closest standard techniques.
So far we have performed T1 measurements on yeasts and macrophages as well as reference samples. In macrophages we were able to follow nitric oxide signaling. There we are able to measure triggering and inhibition of free radical production. We also have already achieved to gain some control over the particle location within the cell by attaching antibodies to the diamond surface. So far co-localisation with the nucleus or with mitochondria has been shown.
In yeast cells we were able to improve diamond uptake and to diffententiate between young and old cells, between different knock-out strains. Additionally, we compared cells that were aged in presence or absence of an antioxidant.
The measurements on test samples helped us to identify which particles and which data analysis method are best to use . Additionally, we could learn from this data what our signals mean.
We have implemented 3D particle tracking was essential record a signal at all from a particle that is moving. However, particle trajectories give interesting additional insights. How fast a particle is moving inside the cell can for instance indicate where within a cell it is. Additionally, one can use the natural movement to obtain information on different parts of the cell. On our way to differentiating between radicals we have adapted the measuring program for pulsing and upgraded the equipment in the group.
For all the experiments that we have done with our technique we have also compared with the closest standard techniques.
While diamond magnetometry has already had some success in the fields of physics, it is rather new in biology. When it comes to work in cells, the only work that has been done before is measuring a cell sample that has been marked with spin labels. In addition to that a different way to use diamond magnetometry has been used to measure temperatures inside cells. Measuring free radicals (or any metabolic activity from a cell) is entirely new.