Researchers have devised a super-resolution microscopy technique that allowed them to observe chemical reactions taking place on the cellular scale, according to a new study in The Journal of Physical Chemistry Letters.
The technique of investigation created in the research makes it possible to see reactions not just inside living cells, but also within individual organelles, like mitochondria.
The chemical mechanisms behind a cell's crucial functions are still relatively mysterious. Only recently, researchers have had the know-how to examine chemical phenomena taking place in living cells directly. However, as a result of technical limitations, researchers still do not have even the most basic knowledge of these reactions. Notably, scientists have been unable to determine how much of a chemical in a given cellular reaction is in a reacted form and how much is in an unreacted form, a ratio known as the equilibrium constant.
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In the new study, scientists from the Institute of Physical Chemistry of the Polish Academy of Sciences (IPC PAS) in Warsaw and the Berlin-based company PicoQuant GmbH created and tested modifications to one of the most modern microscopic methods: super-resolution fluorescence correlation spectroscopy.
In our attempts to determine the equilibrium constants for various reactions in cells, we looked to super-resolution fluorescence correlation spectroscopy. And here we came across an interesting technical problem whose solution opened up new possibilities for us in the study of the chemistry of life.
Robert Holyst, Study Author & Chemistry Professor - IPC PAS
Optical microscopy is the ideal way to examine cells as a result of its low invasiveness and the capability to see spatial structures. For quite a while, its basic disadvantage was its fairly low resolution: fundamental physical constraints (diffraction) make it impossible to differentiate details smaller than approximately 200 nanometres by conventional optical means.
A fairly young branch of optical microscopy known as Fluorescence Correlation Spectroscopy (FCS) is used to analyze the motion of molecules. The technique involves measuring the light emitted by a fluorescent dye connected to the tested molecule that has been excited by a laser beam. Knowing the dimensions of the focus, the period of fluorescence and with the use of theoretical models, it is possible to accurately figure out the velocity of individual molecules, the study team said.
For some time it has been known that while super-resolution FCS microscopy works well when observing molecules moving in two dimensions, e.g., in lipid membranes, it fails in observations in volumes. Diffusion times, determined on the basis of measurements in 3D, could differ from the predictions from measurements in 2D by an order of magnitude or even more. After a few months of research it became clear to us that for these discrepancies were due to the excessively simplified manner of determining the spatial size of the focus.
Krzysztof Sozanski, Study Author - IPC PAS
To reach its conclusion, the study team developed a new theoretical model that adjusted the spatial shape of the focus and took into account its affect on the measured signal-to-noise ratio. The validity of the model was initially established in measurements of the diffusion rate of numerous fluorescent probes in solutions, according to the study.
We also carried out more advanced experiments. For example, we studied a reversible reaction in which the dye molecules attached themselves to micelles and then detached themselves after some time. The system, composed of relatively large balls of surfactant molecules reacting with the molecules of dye, reflected conditions characteristic of biological structures.
Xuzhu Zhang, Co-Author and PhD student at IPC PAS
The observations in these advanced experiments were challenging, the researchers said. If the molecules were moving slowly through the focus, the dye could repeatedly and quickly connect and disconnect and the emitted light would be averaged. However, connection and disconnection reactions could happen so slowly transition through the focus there was no difference in the connection between the reagents, and no averaging would be done.
"Our model takes into consideration not only both of the extreme cases, but also all the intermediate ones,” Zhang said. “And with the knowledge at our disposal about the actual size of the focus, we are able to change its size and experimentally examine all the cases required by the model both in the same chemical system and on the same equipment.”
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