Rotating Laser Enables Faster, Longer Imaging of Cells

A microscopy method developed at the University of Freiburg is able to resolve cellular-level detail without fluorescence, enabling observations 100 to 1000× 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 lightwaves to scatter at the structures of cells to generate images.

“We are exploiting several physical phenomena familiar from everyday life,” said Alexander Rohrbach, a professor at the University of Freiburg. “First, small objects like molecules, viruses, or cell structures scatter — or distribute — blue light the most, which is known from the air molecules in the atmosphere and that we perceive as blue sky.”

Small objects scatter and direct roughly 10× more blue than red light particles to the camera and thereby transmit valuable information.

In a microscopy imaging method developed by researchers at the University of Freiburg, blue laser beams rotate around an object 100× per second (scheme left). The lightwaves scatter at the cell structures (cell) and thus generate 100 superresolved images per second. Within a 10-ms rotation (0° to 360°), continuously deformed lightwaves produce the razor-sharp image of a cell purely from scattered laser light, as shown in the photo. Courtesy of Alexander Rohrbach, University of Freiburg.

The method directs a blue laser at a highly oblique angle on the biological objects, as this markedly 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 illumination from only one direction would produce a great deal of artifacts.

The researchers then rotate the oblique laser beam 100× per second around the object, thereby producing 100 images per second.

“So in 10 minutes, we already have 60,000 image of living cells, which turn out to be far more dynamic than previously thought,” Rohrbach said.

Dynamic analysis like this demands enormous computing power to prove even one minute of visual material, however. Therefore, a variety of computer algorithms and analytical processes first had to be developed so that the data could be properly interpreted.

With colleague Felix Jünger and in cooperation with Freiburg research groups, Rohrbach demonstrated the capacity of the microscope using various cell systems. “Our primary aim wasn’t to generate pretty pictures or films of the unexpectedly high dynamic of cells — we wanted to gain new biological insights,” Rohrbach said.

For instance, the ROCS technology enabled them to observe how mast cells open small pores in just a few milliseconds when stimulated, in order to eject spherical granules at an inexplicably high force and speed. The granules contain the transmitter histamine, which can produce allergic reactions.

In another series of experiments, the team observed how tiny virus-size particles dance in incredible speed around the rugged surface of scavenger cells, taking several tries to find a binding point on the cell. These observations are pretests for currently running studies about the binding behavior of coronaviruses.

Additionally, the ROCS technology has been used within the collaborative research cluster CRC 1425 about the formation of scars in cardiac lesions. Fibroblasts (scar tissue cells) form 100-nm thin tubes. Using the nonfluorescence technology, Jünger and Rohrbach discovered that the tubes vibrate thermally on a millisecond scale and that motion wanes over time. According to mathematical analyses of activity, this indicates a mechanical stiffening of the nanotubes.

The research was published in Nature Communications (www.doi.org/10.1038/s41467-022-29091-0).