FREIBURG, Germany, April 20, 2022 – A microscopy method developed at the University of Freiburg is able to resolve detail at the cellular level without fluorescence, enabling observations 100 to 1000 times longer and 10 to 100 times faster, with almost double the resolution. The technique is called rotating coherent scattering (ROCS). It uses a rapidly rotating blue laser beam, causing light waves to scatter over cell structures to generate images.
“We exploit several physical phenomena familiar from everyday life,” said Alexander Rohrbach, professor at the University of Fribourg. “First, small objects like molecules, viruses or cellular structures scatter – or distribute – the most blue light, which is known from air molecules in the atmosphere and which we perceive as blue skies.”
Small objects scatter and direct about 10 times more blue light particles than red light particles towards the camera and thus transmit valuable information.
In a microscopy imaging method developed by researchers at the University of Fribourg, blue laser beams rotate around an object 100 times per second (left diagram). The light waves diffuse at the level of the cellular structures (cell) and thus generate 100 super-resolution images per second. In a rotation of 10 ms (0° to 360°), continuously distorted light waves produce the razor-sharp image of a cell solely from scattered laser light, as shown in the photo. Courtesy of Alexander Rohrbach, University of Fribourg.
The method directs a blue laser at a very oblique angle at biological objects, as this greatly increases contrast and resolution in a manner similar to how fingerprints on a glass are easier to see when the glass is held at an angle to the light. The scientists illuminate the object successively from each direction with the oblique laser beam because illuminating only one direction would produce a lot of artifacts.
The researchers then rotate the oblique laser beam 100× per second around the object, producing 100 frames per second.
“So in 10 minutes we already have 60,000 live cell images, which turn out to be much more dynamic than previously thought,” Rohrbach said.
However, dynamic analysis like this requires enormous computing power to prove even a minute of visual material. Therefore, a variety of computer algorithms and analytical processes first had to be developed so that the data could be correctly interpreted.
Together with his colleague Felix Jünger and in cooperation with research groups from Freiburg, Rohrbach demonstrated the ability of the microscope to use various cell systems. “Our main goal was not to generate pretty pictures or movies of the surprisingly high dynamics of cells – we wanted to gain new biological insights,” Rohrbach said.
For example, ROCS technology allowed them to observe how mast cells open small pores in just milliseconds when stimulated to eject spherical granules with inexplicably high force and speed. The granules contain histamine, a transmitter which can cause allergic reactions.
In another set of experiments, the team observed how tiny virus-sized particles danced with incredible speed around the rough surface of scavenger cells, taking several tries to find a binding point on the cell. . These observations are pretests for ongoing studies of coronavirus binding behavior.
In addition, ROCS technology has been used within the CRC 1425 collaborative research cluster on scar formation in cardiac lesions. Fibroblasts (scar tissue cells) form 100 nm thin tubes. Using fluorescence-free technology, Jünger and Rohrbach found that the tubes vibrated thermally on a millisecond scale and the motion decreased over time. According to mathematical analyzes of activity, this indicates a mechanical stiffening of the nanotubes.
The research has been published in Nature Communication (www.doi.org/10.1038/s41467-022-29091-0).