Rotating laser light illuminates living cells

Friday, 22 April, 2022

Rotating laser light illuminates living cells

In fluorescence microscopy, biological cells are marked with fluorescent dyes and excited to luminesce in specific areas via optical switches. However, this light is usually too faint for small, rapid objects, or even goes out after a while. Now Prof Dr Alexander Rohrbach and his team at the University of Freiburg have found a way to make the smallest objects clearly visible without fluorescence, thanks to a process called ‘rotating coherent scattering’ (ROCS) that uses a rapidly rotating blue laser beam.

While fluorescence microscopy records what you might call ‘night-time images’ of structures, ROCS microscopy takes ‘daytime images’ — opposites that can complement each other admirably. Furthermore, cellular structures or virus-sized particles can be observed 100 to 1000 times longer, 10 to 100 times faster and with almost doubled resolution than with fluorescence microscopy. The technology has been described in the journal Nature Communications.

“We are exploiting several physical phenomena familiar from everyday life,” Rohrbach said. “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 times more blue than red light particles to the camera and thereby transmit valuable information.

ROCS targets a blue laser at a highly oblique angle on the biological objects, because this markedly increases contrast and resolution. The scientists also illuminate the object successively from each direction with the oblique laser beam, because illumination from only one direction would produce a lot of artefacts.

The researchers rotate the oblique laser beam 100 times per second around the object and thereby produce 100 images per second — “so in 10 minutes we already have 60,000 images of living cells, which turn out to be far more dynamic than previously thought,” Rohrbach said. Dynamic analyses like this demand enormous computing power, so a variety of computer algorithms and analytical processes were developed so that the data could be properly interpreted.

Rohrbach was able to demonstrate the capacity of the microscope using various cell systems. For instance, the technology enabled him 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 subsequently lead to allergic reactions.

In another series of experiments, the researchers were able to observe how tiny virus-sized 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 served as pretests for current studies about the binding behaviour of coronaviruses.

The technology has also been used to look at the formation of scars in cardiac lesions. The researchers found that scar tissue cells form 100 nm-thin nanotubes which vibrate thermally on a millisecond scale, but that this motion wanes over time, indicating a mechanical stiffening of the nanotubes. Finally, the scientists were able to observe over many thousands of images how filopodia — the ‘fingers’ of scavenger cells — search their environment for prey using a complex dither movement and how filopodia can alter their cytoskeleton at previously unknown speeds.

Image credit: ©ürgen Fälchle

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