A super-resolution view of chemical reactions

February 12, 2018, Polish Academy of Sciences
The new method of data analysis from super resolution fluorescence correlation microscopy has been verified, among others, in experiments that imitate the biological environment. Researchers observed small fluorescent dye molecules that attach and detach from/to relatively large, spherical micelles. Credit: IPC PAS, Grzegorz Krzyzewski

Researchers from the Institute of Physical Chemistry of the Polish Academy of Sciences have demonstrated, using a super-resolution microscopic technique, how to follow chemical reactions taking place in very small volumes. The method was developed in collaboration with PicoQuant GmbH, and makes it possible to observe reactions within individual cellular organelles such as cell nuclei.

The chemical mechanisms responsible for the cell's vital functions still conceal many secrets—only recently have researchers had the tools to look directly at the chemical phenomena occurring in living cells. However, due to continuing technical limitations, science lacks basic knowledge about the equilibrium constant values of chemical reactions in cells. In other words, researchers still do not know how much of a chemical involved in a given cellular is in an already reacted form and how much is in an unreacted form. These challenges have been overcome in the current study. The research collaborative has developed and demonstrated a modification of super-resolution fluorescence correlation spectroscopy.

"We have been dealing with 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 in situations involving low concentrations of reagents," says Prof. Robert Holyst (IPC PAS). "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 . 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."

There are many varieties of microscopy, including those that visualize individual atoms. However, when observing cells, optical microscopy remains unbeatable due to its low invasiveness and the ability to visualize the spatial structure of living organisms. For a long time, its basic disadvantage was its relatively poor resolution—fundamental physical constraints (diffraction) make it impossible to distinguish details smaller than about 200 nanometres by standard optical techniques.

One type of optical microscopy is . It involves introducing a fluorescent dye into the sites of the biological sample being studied, and then scanning the sample with a focused laser beam, which stimulates the dye molecules to glow. In 1994, Stefan W. Hell presented a method of exceeding the diffraction limit in fluorescence microscopy by means of stimulated emission depletion (STED). STED requires an additional laser beam resembling a doughnut in cross-section. This beam extinguishes the external areas of the main focus of the laser beam and consequently reduces its size to values below the diffraction limit. With super-resolution methods it is now possible to see spatial details of only 10 nm with a time resolution of up to microseconds.

At the Institute of Physical Chemistry of the Polish Academy of Sciences in Warsaw, it was shown how to observe the course of chemical reactions in extremely small volumes, comparable to the size of cell nuclei, by means of super resolution fluorescence correlation microscopy. In the picture, doctoral student Xuzhu Zhang in the laboratory Credit: IPC PAS, Grzegorz Krzyzewski

Fluorescence correlation spectroscopy (FCS) is a new branch of for studying the motion of molecules. In super-resolution varieties, the focus of the laser has a volume measured in tens of attolitres (one attolitre is a billionth of a billionth of a litre). The measurement involves measuring the light emitted by a attached to the tested molecule excited by a . Knowing the size of the focus and the duration of fluorescence, and with the assistance of the appropriate theoretical models, it is possible to determine the velocity of even individual molecules.

"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 3-D, could differ from the predictions from measurements in 2-D by an order of magnitude or even more. After a few months of research, it became clear to us that these discrepancies were due to the excessively simplified manner of determining the spatial size of the focus," says Dr. Krzysztof Sozanski (IPC PAS).

On the basis of their own theoretical analyses and experiences, the Warsaw researchers constructed a new, universal theoretical model introducing a correction of the spatial shape of the focus and taking into account its impact on the measured signal-to-noise ratio. The correctness of the model was initially verified in measurements of the diffusion rate of various fluorescent probes in solutions.

"We also carried out more advanced experiments. For example, we studied a reversible reaction in which the 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," says Ph.D. student Xuzhu Zhang (IPC PAS). The measurements were not simple. If the molecules of both reactants were moving slowly, when passing through the focus the dye could repeatedly merge/disconnect with/from the micelles and the emitted light would be averaged.

But there could also be a variant of the other extreme: the connection and disconnection reactions could run so slowly that during the transition through the focus there would be no change in the relationship between the reagents—then there would be no averaging. "Our model takes into consideration not only both of the extreme cases, but also all the intermediate ones. 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," emphasizes Zhang.

An important feature of the analytical method developed at the IPC PAS is the fact that no changes in apparatus are needed for its application. After appropriate adaptation, the method can be used to more accurately interpret data recorded by FCS-ready STED microscopes already in production.

Explore further: Optical nanoscope images quantum dots

More information: Xuzhu Zhang et al, Nanoscopic Approach to Quantification of Equilibrium and Rate Constants of Complex Formation at Single-Molecule Level, The Journal of Physical Chemistry Letters (2017). DOI: 10.1021/acs.jpclett.7b02742

Related Stories

Optical nanoscope images quantum dots

January 23, 2018

Physicists have developed a technique based on optical microscopy that can be used to create images of atoms on the nanoscale. In particular, the new method allows the imaging of quantum dots in a semiconductor chip. Together ...

Background suppression for super-resolution light microscopy

February 1, 2017

Researchers of Karlsruhe Institute of Technology (KIT) have developed a new fluorescence microscopy method: STEDD (Stimulation Emission Double Depletion) nanoscopy produces images of highest resolution with suppressed background. ...

New dye allows super-imaging of cells

April 11, 2017

A new dye might allow researchers to view natural processes in extremely small components of living cells over a prolonged period of time; a previously unattainable feat.

Recommended for you

Nanodiamonds as photocatalysts

October 19, 2018

Climate change is in full swing and will continue unabated as long as CO2 emissions continue. One possible solution is to return CO2 to the energy cycle: CO2 could be processed with water into methanol, a fuel that can be ...

Producing defectless metal crystals of unprecedented size

October 19, 2018

A research group at the Center for Multidimensional Carbon Materials, within the Institute for Basic Science (IBS), has published an article in Science describing a new method to convert inexpensive polycrystalline metal ...

Shining light on the separation of rare earth metals

October 18, 2018

Inside smartphones and computer displays are metals known as the rare earths. Mining and purifying these metals involves waste- and energy-intense processes. Better processes are needed. Previous work has shown that specific ...


Please sign in to add a comment. Registration is free, and takes less than a minute. Read more

Click here to reset your password.
Sign in to get notified via email when new comments are made.