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