Rotating blue laser light reveals unimagined dynamics in living cells


Date:
April 13, 2022
Source:
University of Freiburg
Summary:
When cities transform into a colorful world of lights as darkness
falls, it's often only possible to estimate their contours, which
depending on the perspective can draw the attention to key details
or trivia. In fluorescence microscopy, biological cells are marked
with fluorescent dyes and excited to luminesce in specific areas
by optical switches- like a city at night. However, this light is
usually too faint for small, rapid objects, or even goes out after a
while. This is known as 'fluorescence bleaching.' Now, researchers
have developed a new way to make the smallest objects clearly
visible without fluorescence. In this way, cellular structures or
virus-sized particles can be observed 100 to 1,000 times longer,
ten to 100-times faster and with almost doubled resolution than
with fluorescence microscopy.



FULL STORY ==========================================================================
When cities transform into a colorful world of lights as darkness
falls, it's often only possible to estimate their contours, which
depending on the perspective can draw the attention to key details or
trivia. In fluorescence microscopy, biological cells are marked with fluorescent dyes and excited to luminesce in specific areas by optical switches- like a city at night. However, this light is usually too
faint for small, rapid objects, or even goes out after a while. This
is known as " fluorescence bleaching." Now, a new approach developed
by Prof. Dr. Alexander Rohrbach and his team in the Laboratory for Bio-
and Nano-Photonics at the University of Freiburg has found a way to make
the smallest objects clearly visible without fluorescence. In this way, cellular structures or virus-sized particles can be observed 100 to 1,000
times longer, ten to 100-times faster and with almost doubled resolution
than with fluorescence microscopy. While fluorescence microscopy records
what you might call "night-time images" of structures, ROCS microscopy
takes "day-time images" -- opposites that can complement each other excellently. Rohrbach and his colleagues describe various applications
of the technology in the latest issue of Nature Communications.


==========================================================================
Blue laser directed illuminates object at oblique angle The technology
they use is known as 'Rotating Coherent Scattering' (ROCS) and uses a rapidly-rotating blue laser beam. "We are exploiting several physical
phenomena familiar from everyday life," explains Rohrbach, "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 ten-times more blue than red light
particles to the camera and thereby transmit valuable information.

Secondly, ROCS targets a blue laser at a highly oblique angle on
the biological objects, because this markedly increases contrast
and resolution. This is familiar to us already as well: if you hold
a wine glass at an angle to the light it's far easier to spot dirt or fingerprints. Thirdly, the scientists illuminate the object successively
from each direction with the oblique laser beam, because illumination
from only one direction would produce a lot of artifacts.

100 images per second of living cells The Freiburg physicist and
engineers from the Department of Microsystems Engineering (IMTEK)
rotate the oblique laser beam a hundred times per second around the
object and thereby produce 100 images per second. "So in ten minutes
we already have 60,000 images of living cells, which turn out to be far
more dynamic than previously thought," says Rohrbach. Dynamic analyses
like this demand enormous computing power to process just 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.

Together with his colleague Dr. Felix Ju"nger and in cooperation with
various Freiburg research groups, Rohrbach was able to demonstrate 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." 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 subsequently lead
to allergic reactions.

Observing thebinding behavior of virus-sized particles 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 currently running studies
about the binding behavior of coronaviruses.

In addition, the ROCS technology has been used within the collaborative research cluster CRC 1425 about the formation of scars in cardiac lesions.

Fibroblasts, that is scar tissue cells, form 100 nanometers thin tubes,
so- called nano-tubes, which are 1,000-times thinner than a hair. By
this new technology Ju"nger and Rohrbach were able to discover that
these tubes vibrate thermally on a milliseconds scale, but this motion
wanes over time. According to mathematical analyses of activity, this
indicates a mechanical stiffening of the nano-tubes.

In other experiments the scientists were finally 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.


========================================================================== Story Source: Materials provided by University_of_Freiburg. Note:
Content may be edited for style and length.


========================================================================== Related Multimedia:
*
Illustration_of_blue_laser_beams_that_rotate_around_the_object_100_times
per_second ========================================================================== Journal Reference:
1. Felix Ju"nger, Dominic Ruh, Dominik Strobel, Rebecca Michiels,
Dominik
Huber, Annette Brandel, Josef Madl, Alina Gavrilov, Michael
Mihlan, Caterina Cora Daller, Eva A. Rog-Zielinska, Winfried
Ro"mer, Tim La"mmermann, Alexander Rohrbach. 100 Hz ROCS microscopy
correlated with fluorescence reveals cellular dynamics on different
spatiotemporal scales. Nature Communications, 2022; 13 (1) DOI:
10.1038/s41467-022- 29091-0 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2022/04/220413161812.htm

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