Track and trace method predicts best possible resolution in microscopy

TU Delft scientists provide insight into the limits of super-resolution microscopy and propose a new calculation method for determining the maximum resolution. Technology is important for studying processes in the living cell, discovering the origin of diseases and developing new drugs. Their findings were published in the Biophysical Journal.

In 2019, researchers from Delft had already given themselves the field of super-resolution microscopy a considerable boost in improving the accuracy of the technique about double. Now they have published a scientific paper that points 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, are laying new groundwork for the super-resolution method called iterative single-molecule localization microscopy. They use lighting patterns to zoom in on molecules. To do this, they use the results of previous experiments to bring the patterns of the molecules closer and closer. This increases the sharpness of the image exactly where the molecules are.

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

Super resolution microscopy

Super-resolution microscopy is a breakthrough technology that allows researchers to look inside living cells. The technique uses luminescent proteins found, for example, in jellyfish. In 2008, three top researchers received 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) ensures that molecules are turned on or off randomly. Sensitive sensors make a video of these light signals, after which the researchers analyze the data obtained. This allows them to very precisely determine the location of molecules and to reconstruct the cell structure. With an ordinary optical microscope, you can make images at a scale of about half a micron. Super-resolution microscopy increases this capacity by a factor of ten.

Development of super-resolution microscopy

The field of super-resolution microscopy has grown 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 the German researcher Stefan Hell. Hell’s lab researchers argued in 2020 that iterative single-molecule localization microscopy would significantly improve resolution. TU Delft scientists now show that these major resolution improvements are virtually unattainable in practice.

Kalisvaart: “In a practical situation, the best you can hope for is about a fivefold improvement over standard technique. The field has widely assumed there is much greater potential. We have now looked at this problem for the first time using a different mathematical (Bayesian) approach and showed that the improvements in resolution of Hell’s group are difficult to achieve in practice.”

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