As biotechnology and nanotechnology become increasingly prevalent in our lives, an understanding of the fundamental biology is only growing more important. As well as the implications for medicine, evolution has had billions of years to optimize for the transport and storage of energy, or molecules: processes that are of great interest across many technologies. But until recently, progress in this field had been limited by a fundamental deficiency: our resolution of chemical reactions inside the cell was severely limited. However, this is starting to change.
New research from the Institute of Physical Chemistry of the Polish Academy of Sciences uses a super-resolution microscopic technique that can allow chemical reactions to be tracked even if they involve minimal volumes of reagent. This makes it possible not just to observe chemical reactions that occur within the cell, but also within individual organelles such as the nucleus, mitochondria, or chloroplasts. This could unlock information about how cells are controlled, or about processes that humans are trying to synthetically mimic, like photosynthesis.
The potential industrial applications of this fundamental research are reflected in the fact that it was aided by a collaboration between the IPC PAS researchers and a Berlin-based company, PicoQuant GmbH, a photonics company that specializes in high-resolution spectroscopy, data analysis, and collection. The technique that is used is called super-resolution fluorescence microscopy. Optical microscopy has often been preferred in the study of delicate biological systems, as it’s non-invasive and non-destructive, allowing you to observe natural processes in the cell as well as visualizing its structure.
We have been dealing with chemical reactions in cells for a long time. For example, in 2013, we determined the diffusion coefficients of all the proteins in the Escherichia coli bacterium, thanks to which it became possible to determine the rate of reactions taking place with their participation. Here we were interested in a similar issue, but with regard to the situation when we have low concentrations of reagents. Biological reactions are generally reversible and, where they occur, a certain dynamic equilibrium is usually created between the amount of reacted and unreacted substances. 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.
Professor Robert Holyst
The main issue with optical microscopy is down to the fundamental physics: diffraction-limited resolution means that typically one cannot resolve on scales less than 200nm with standard optical techniques. This research uses a method that smashes through the diffraction barrier, allowing for resolution of features around 10nm in length, and time-resolution on the order of microseconds: on the relevant time and length-scales for individual reactions in the organelles.
The technique builds on standard fluorescence microscopy; in this procedure, a fluorescent dye is injected into the relevant parts of the biological sample, which is then scanned with a laser. By measuring the fluorescing dye, the structure of the specimen can be determined. Typically, that laser-beam would diffract to a size of a few hundred nanometers: you could only resolve features of this scale or larger. Since the 1990s, a technique called Stimulated Emission Depletion – which uses an additional beam to extinguish the areas of external focus of the main laser-beam using interference – can reduce the size of that laser-beam to smaller than the diffraction limit.
Fluorescence correlation spectroscopy (FCS) – which involves attaching a fluorescent dye to an individual molecule – can be used to measure particles moving through fluids. This can include stunningly high-resolution – perhaps down to a few tens of attolitres, where an attolitre is 10-18 liters. But measuring exact diffusion times with this new technique had previously been difficult: "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," says Dr. Krzysztof Sozanski (IPC PAS).
The Warsaw researchers have created a new theoretical model that can convert these measurements into diffusion rates for the various molecules. By taking into account the impact of the spatial shape of the beam focus on signal-to-noise ratio and diffusion time measurements, they are now able to correct these measurements. Their model was validated by tests on fluorescent probes in many different solutions.
Their theoretical model can now accurately measure diffusion times for molecules in 3D, and even account for extreme cases where the fluorescent dye molecule regularly detaches and recombines with the molecule that the team is attempting to trace. The team is hopeful that these measurements, improved in reliability and resolution, will be able to enhance our understanding of fundamental biological processes – and perhaps harness those secrets ourselves.