Track-and-trace method predicts the best possible resolution in microscopy

Biophysical Journal (2022). DOI: 10.116/j.bpj.2022.05.027″ width=”753″ height=”479″/>

Pattern position control strategy (see equation 5) to iteratively increase the information content of signal photons. (a) Example of a period of a sinusoidal intensity pattern in the x direction, with the intensity minimum centered on a global phase zero. (b) Individual illumination patterns placed during iteration 2. In each iteration, four sinusoidal illumination patterns are placed so that the current estimate of the emitter position is sandwiched between the illumination minima of the patterns. (c) Summed illumination patterns over the course of three iterations. The distance between the intensity minimum of the summed patterns and the transmitter position becomes iteratively smaller as a result of adjusting the search area based on previous information about the precision achieved in the previous iteration. (d) Expected signal photon response from the emitter in (b) and (c) over the course of three iterations, using the illumination placement of (c). (e) Illustration of the expected signal photon budget for one, two and three iterations. This article considers two scenarios, namely the case where the number of signal photons is kept constant over the course of all iterations and the case where the imaging time and illumination intensity are kept constant over the course of all iterations. In the latter case, the signal photon budget is depleted by imeSMLM only in the event that the single emitter is illuminated at maximum intensity during all iterations. If the intensity pattern minima are placed close to the emitter, a reduced number of photons are registered within the same time window. Credit: Biophysical Journal (2022). DOI: 10.116/j.bpj.2022.05.027

Scientists from TU Delft provide insight into the limitations of super-resolution microscopy and offer a new calculation method to determine the maximum resolution. The technology is important for studying processes in the living cell, discovering the origin of diseases and developing new medicines. Their findings were published in the Biophysical Journal

In 2019, Delft researchers had already given the field super resolution microscopy a significant boost by approximately doubling the precision of the technique. Now they have published a scientific paper pointing out the fundamental limitations of super-resolution microscopy. “We also provide a method for other researchers to help them make more informed choices,” says Delft Ph.D. student and first author of the publication, Dylan Kalisvaart.

The researchers, led by Carlas Smith, laid a new foundation for the super-resolution method called iterative single-molecule localization microscopy. They use lighting patterns to zoom in on individual molecules† To do this, they use results from previous experiments to move the patterns closer and closer to molecules. This makes it possible to increase the sharpness of the image exactly where the molecules are located.

Kalisvaart, researcher at the Delft Center for Systems and Control, explains: “We show (with the so-called Van Trees inequality) that resolution improvements can be attributed to prior knowledge obtained from previous experiments. This allows us to show what the practical settings of a microscope should be, given the circumstances and prior knowledge, to achieve the best result.”

Super-resolution microscopy

Super-resolution microscopy is a groundbreaking technology that allows researchers to look inside living cells. The technique uses light-emitting proteins that occur in jellyfish, for example. In 2008, three top researchers were awarded the Nobel Prize in Chemistry for the discovery and development of this luminescent protein, called GFP (Green Fluorescent Protein). Researchers can attach these fluorescent proteins to molecules using gene editing. When you shine a laser on these proteins, they emit a small amount of light.

Single molecule localization microscopy (SMLM) allows molecules to be switched on or off at random. Sensitive sensors make a video of these light signals, after which researchers analyze the data obtained. This allows them to very precisely determine the location of the molecules and make a reconstruction of the cell structure. With an ordinary optical microscope you can make images on a scale of about half a micron. Super-resolution microscopy increases this ability by a factor of ten.

Development of super-resolution microscopy

The field of super-resolution microscopy has developed rapidly over the past decade. In 2014, three researchers were awarded the Nobel Prize in Chemistry for what became known as super-resolution microscopy. One of the three winners was German researcher Stefan Hell. Researchers at Hell’s lab argued in 2020 that iterative single-molecule localization microscopy would vastly improve resolution. The scientists at TU Delft have now shown that these large resolution improvements are virtually unattainable in practice.

Kalisvaart: “In practice, you can best hope for an improvement of about five times over the standard technique. The field largely assumed that there was much more potential. We have now looked at this problem for the first time with another mathematical (Bayesian) approach and have shown that Hell’s Group’s resolution improvements are difficult to achieve in practice.”

Will people see the publication in Biophysical Journal especially as a setback? “I don’t see it that way,” says Carlas Smith, Kalisvaart supervisor. “It’s essential that the underlying science is solid. If something is wrong with the structure, you have to go back to ground level and rebuild the foundation.”


Combination of microscopy techniques makes images twice as sharp


More information:
Dylan Kalisvaart et al, Precision in iterative modulation enhanced single-molecule localization microscopy, Biophysical Journal (2022). DOI: 10.116/j.bpj.2022.05.027

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