Super-resolution microscopes achieve nanometer resolution without traditional on/off switching

Overcoming resolution limitations with light microscopes at approximately half the wavelength of light (approximately 250 nanometers) is one of the most important developments in optics. Due to the nature of the light waves, even the best lenses cannot produce light spots smaller than 250 nanometers in diameter. All the molecules in this bright spot are illuminated at the same time and lit together, making it seem inseparable as a blurred whole.

In the early 1990s, Stefan Hell realized that molecules could be separated by switching the molecular signal “off” and “on” so that closely adjacent molecules were forced to signal continuously. Numerators that send continuous signals can be easily distinguished.

Fluorescence microscopy allows for easy turn on and off molecular fluorescence, allowing perfect implementation of this on/off separation principle. In fact, Sted and Palm/Storm, and the recent super-lytic fluorescence microscopes, are all based on this on/off principle.

It temporarily transferred nearby molecules from the fluorescence to the non-fluorescent state, and vice versa, similarly, to the basis of the rapid growth field of super-lytic fluorescence microscopy. In 2014, Hell and American scientists Eric Betsig and William E. Morner were awarded the Nobel Prize in Chemistry for the development of ultra-lytic fluorescence microscopes.

Super resolution with no on/off

Göttingen’s team of scientists led by Hell of the Max Planck Institute for Interdisciplinary Science (MPI) and Heidelberg’s MPI for Medical Research currently demonstrate that castal number molecules can be separated without on/off switching. Their research has been published in the journal Nature Physics.

Researchers experimentally demonstrated that point objects such as molecules can be clearly separated to a small distance of 8 nanometers. To do this, they scanned the molecules with a beam of light, characterized by a zero intensity line (node) in the center. When the beam scans across the sample, the measured signal is registered. For fluorescent molecules, the signal is fluorescence.

For a single fluorescent molecule, the signal is zero only if the zero intensity node of the illumination beam matches the position of the molecule. However, if the sample contains more than one adjacent molecule, the measured signal will not be zero. This is because at least one molecule cannot match the zero intensity point of illumination light anyway. Thus, the position of the molecule is encoded to the deviation of the measured signal from zero.

Scientists have demonstrated that molecular locations can be determined with a very accurate theory and experimentally using this principle of “scanning at minimum intensity.” For example, they were able to separate two permanently ejecting fluorophores at a distance of up to 8 nanometers. We also resolved groups of three or four molecules at a distance of about 20 nanometers.

“On/Off switching has been considered a necessary prerequisite for high optical resolution since we introduced the STED principle for blocking fluorescence over 30 years ago. The concept of minimizing constantly ejected molecules is a breakthrough,” explains Hell.

Thomas Hensel, the first author of the study and a student on the PhD Hell’s team, said, “This new concept makes it easier, in principle, to spatially register molecules closer than those that are even further apart. This is not clear.

Previously, the closer the molecules were to each other, the more difficult it was to solve them. When separating molecules using bright light spots and generated signal maximum values, as in the past, it is difficult to distinguish them due to the signal-to-noise ratio of each molecule.

“If you work on a dark spot or node and look at the deviation of the signal from scratch, that’s actually the opposite,” adds Hell.

Imaging of waves without resolution limits

Scientists at Max Planck believe their results have great potential.

“The idea of ​​minimal decomposition applies not only to fluorescent molecules, but also to any molecule that generally provides a contrasting signal. It applies not only to light waves such as light, but to all kinds of waves,” says Hell.

“It is important to resolve at a minimum distance that is not on or off, to allow for continuous observation of all molecules. There is no interruption by the need to turn off the molecule.”

Continuous observation opens up further applications. If molecular machines such as proteins and protein complexes are constantly labeled at various points with signaling molecules, they should be able to track the changes in fine positions so that they can “take” the actual work of these nano-sized machines of life.

In the future, this will help you design drugs that prevent or support specific proteins to perform their functions, if necessary. By providing insight into how proteins function mechanically, this microscopy could ultimately speed up drug discovery.