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Most optical technologies are applied to flat, basically two-dimensional cellular systems. However, physiological
meaningful information relies on the morphology, the mechanical properties and the biochemistry of a cell's
context. A cell requires the complex three-dimensional relationship to other cells. However, the observation of
multi-cellular biological specimens remains a challenge. Specimens scatter and absorb light, thus, the delivery of
the probing light and the collection of the signal light become inefficient; many endogenous biochemical
compounds also absorb light and suffer degradation of some sort (photo-toxicity), which induces malfunction of a
specimen. In conventional and confocal fluorescence microscopy, whenever a single plane, the entire specimen is
illuminated. Recording stacks of images along the optical Z-axis thus illuminates the entire specimen once for
each plane. Hence, cells are illuminated 10-20 and fish 100-300 times more often than they are observed. This
can be avoided by changing the optical arrangement. The basic idea is to use light sheets, which are fed into the
specimen from the side and overlap with the focal plane of a wide-field fluorescence microscope. In contrast to
an epi-fluorescence arrangement, such an azimuthal fluorescence arrangement uses two independently operated
lenses for illumination and detection. Optical sectioning and no photo-toxic damage or photo-bleaching outside a
small volume close to the focal plane are intrinsic properties. Light sheet-based fluorescence microscopy (LSFM)
takes advantage of modern camera technologies. LSFM can be operated with laser cutters and for fluorescence
correlation spectroscopy. During the last few years, LSFM was used to record zebrafish development from the
early 32-cell stage until late neurulation with sub-cellular resolution and short sampling periods (60-90 sec/stack).
The recording speed was five 4-Megapixel large frames/sec with a dynamic range of 12-14 bit. We followed cell
movements during gastrulation, revealed the development during cell migration processes and showed that an
LSFM exposes an embryo to 200 times less energy than a conventional and 5,000 times less energy than a
confocal fluorescence microscope. Most recently, we implemented incoherent structured illumination in our
DSLM. The intensity modulated light sheets can be generated with dynamic frequencies and allow us to estimate
the effect of the specimen on the image formation process at various depths in objects of different age.
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