Under green fluorescent light, cell structures, here microtubuli, can be observed in living fish embryos. Figure: NIH, KIT |
Microscopes
provide valuable insights in the structure and dynamics of cells, in
particular when the latter remain in their natural environment. However,
this is very difficult especially for higher organisms. Researchers of
Karlsruhe Institute of Technology (KIT), the Max Planck Institute for
Polymer Research, Mainz, and the American National Institutes of Health
(NIH) have now developed a new method to visualize cell structures of an
eighth of a micrometer in size in living fish larvae. It is published
in Nature Methods.
“The
zebrafish is perfectly suited for genetic studies of cells, as its
larvae are completely transparent,” explains Marina Mione, KIT. To
visualize certain structures, these are colored mostly by genetic
engineering methods using a fluorescent dye. Mione studied parts of the
cellular skeleton of fish, the so-called microtubuli. The thread-shaped
microtubuli have a length of about 100 µm and a diameter of about 20 nm,
corresponding to a hundred thousandth of a human hair. “Microtubuli
exist everywhere in the cell and are required for its division and
motion.”
In
the new microscopy method, the object is not illuminated completely,
but only at a certain spot with special light. Scattered light is
minimized and the illuminated detail is represented sharply. A series of
images taken at variable illumination is then processed by a computer.
In this way, an overall image is obtained. Smart illumination even
allows to adjust the depth of field, to image various depth levels, and
to combine them into a three-dimensional image on the computer.
“Meanwhile,
it is possible to reach resolutions of 145 nm in the plane and 400 nm
in-between,” says Marina Mione. The images are taken within a few
seconds, such that movement of the cells does not cause any unsharpness.
Based
on a series of images, videos of the movement of the microtubuli are
obtained. In the experiment, it was observed over a period of 60 minutes
how the early stage of the fish’s lateral line develops about 45 µm
below the skin of the fish. Via this organ, the fish perceives movement
stimuli in water. Such images of living organisms also provide valuable
findings regarding the development of vertebrates on the cellular level.
The
tropical zebrafish living in freshwater has several advantages as a
genetic model organism. It is sufficiently small for easy cultivation
and large enough to easily distinguish individual organs. It has a short
generation cycle and produces many offsprings. As a vertebrate, it has a
number of microbiological properties in common with human beings.
