Biomedical Engineering Reference
In-Depth Information
from the nerve fibers were found to elongate from the ventricular zone to the outer region, called mar-
ginal zone or white matter, and this also matches the fact that the differentiated ganglia cells elongate
their nerve fibers from the ventricular zone to the marginal zone in the direction perpendicular to the
neural tube (Kimmel et al. 1995). By applying the combined SHG/THG microscope, the mitosis process,
the polarized neuron formation, and the neural stem cell behaviors can thus be dynamically tracked
without the help of fluorescence markers.
In addition to the brain, the eye is also one important part of the peripheral nervous system worth
being studied. By making use of SHG contrasts in nerve fibers and stack membranes, embryonic devel-
opment of the zebrafish eyes can be carried out to see the elongating of the nerve fibers and the matura-
tion of the retina. Meanwhile, the THG modality can help to show the morphological changes during
the development process. At the 14-somite stage, a flat tissue called optic vesicle and a slit called the
optic lumen (Li et al. 2000) in the middle of the optic vesicle can already be observed through the THG
modality (Figure 14.10a), while the SHG signals are originated from mitotic spindles (Figure 14.10b).
At the prim-5 stage, this flat tissue is found to transform into the shape of a cup, called eye cup, and
the lens can be distinguished then. From the prim-12 to the prim-14 stage, the first neuron in retina
is then born, SHG signals are found at the bottom of the eye cup to show the newborn nerve fibers
(Figure 14.10c). After the prim-14 stage, the SHG signals gradually became denser and stronger in the
retina, which showed the elongation and maturation of the nerve fibers. Meanwhile, the outline of the
lens and the retina can all be revealed distinctly by the THG modality. By
in vivo
observing a 60-hpf
embryo with its left side upward and optically sectioning the eyes downward from the lens to the retina
and then the neural tube, the optic nerves, which consist of myelin-sheathed nerve fibers (arrow), is
revealed by the SHG modality extending from the lens to the neural tube (Figure 14.10d). In addition
to the optical nerves, strong SHG signals are also found at the bottom of the retina. In the SHG/THG
image of a paraffin section of a 4-day-embryo eye (Figure 14.10e), the SHG signals are shown to have
good contrast in every layer full of nerve fibers, including inner and outer plexform layers (arrows) and
optical nerve (dotted arrow). Besides, the strong SHG signals at the bottom of the retina are found to
arise from the outer segment of the photoreceptors (arrow). As the same origin for the SHG signals in
grana (Huang et al. 1989), the strong SHG signals observed in the outer segment are the result of the
stacked membranes.
Owing to the least-invasive nature of SHG/THG microscopy, the important morphogenetic changes
of the zebrafish brain from the bud stage to the prim-15 stage were
in vivo
monitored continuously in the
same live embryo without any fluorescence markers. Through the SHG modality, we are able to dynami-
cally record the cell mitosis, cell differentiation, and the nerve fiber developments in the brain and the
retina. Combined with THG modality, more information about the morphological changes during the
development can be obtained and the valuable SHG signals from cell mitosis and nerve fiber develop-
ment can be easily localized and recognized. Therefore, the combined SHG/THG microscopy is shown
to have strong capability for various studies of the nervous system.
14.3.2.3 Somite Development
Based on the strong SHG contrasts provided by muscle fibers that are composed of collaterally orga-
nized myosin and actin filaments and have crystalline nanostructure (Chu et al. 2002), the combined
SHG/THG microscopy can also be applied to studies of embryonic somite development. Since numer-
ous mysteries about the somite development, including pinching of of somites, mytomes development,
and differentiation and migration of muscle fibers, have not been revealed yet, the SHG/THG system
may provide a useful tool for solving those problems. Through SHG modality, the detailed distribu-
tion of muscle fibers in a somite can be observed, where individual sarcomere composed of A- and
I-bands with a periodicity <2 μm inside muscle fibers can be readily resolved; the general morphological
structures, including the boundary of somite and notochord, can be revealed through THG modality.
Starting from about 10.3 hpf, when it is the beginning of the segmentation period, the somites will
appear sequentially in the trunk and tail and provide a staging index of the embryonic development. At
