Combined SHG/THG Imaging - Second Harmonic Generation Imaging - page 320

(arrow in Figure 14.6h), while THG contrasts can be found in adipocytes (arrow in Figure 14.6g) and
RBC (arrowhead in Figure 14.6h). In addition to the animal models, our combined SHG/THG micro-
scopy has been preliminarily applied on excised human tissues like teeth (Chen et al. 2008), lung (Yu
et al. 2007), cartilage (Tsai et al. 2009), and skin (Tai et al. 2005; Chen et al. 2009a). The strain status of
the abnormal enamel (arrow in Figure 14.6i), type II collagen in the cartilage (Figure 14.6k), and collag-
enous structures of dermis (D) of the skin (Figure 14.6l) can be revealed by SHG microscopy, while THG
microscopy was shown to be able to reveal the rod structures of the tooth enamel (Figure 14.6i), elastic
fibers in the lung tissues (arrow in Figure 14.6j), chondrocytes in the cartilage (arrow in Figure 14.6k),
and cellular morphology of epidermis (ED) of the skin (Figure 14.6l). These preliminary studies not only
show the rich imaging contrasts of the combined SHG/THG system, but also indicate the appreciated
imaging capability of the combined SHG/THG system for clinical trails.
14.3.2 Animal Models: Zebrafish
Complex developmental processes of vertebrate embryos are difficult to observe noninvasively with
high penetrability and high spatial resolution at the same time. In our previous in vivo studies (Chu
et al. 2003; Sun et al. 2004; Chen et al. 2006), zebrafish embryos were used for investigating the complex
embryonic development process, since the zebrafish embryos have much genetic material the same as
humans' and have similar but simpler embryonic developmental programs. In addition, rapid devel-
oping rate, precisely defined developing stages, its transparency, small size, and external development
also facilitate the neurology study and microscopic observation. Within the zebrafish embryos, SHG
modality can provide various and valuable information, including mitosis spindle fibers (Chu et al.
2003; Sun et al. 2004; Chen et al. 2006), nerve fibers (Chen et al. 2006), muscle fibers (Chu et al. 2003;
Sun et al. 2004), and stacked membranes (Chen et al. 2006), to study the embryonic development of
different systems and different stages. Combined with THG modality, which can reflect the structural
information of the embryos, can help to localize the SHG signals and identify and contrast sources of
the SHG signals. Owing to embryos' transparency and ~1.5 mm thickness, the SHG/THG microscope
with forward-collection geometry was used for zebrafish embryos' in vivo investigations. In the follow-
ing subsections, previous SHG/THG studies of cell mitosis, nervous system development, and somite
development of the zebrafish embryos will be introduced.
14.3.2.1 cell Mitosis
In the in vivo investigation of cell mitosis in zebrafish embryos at 1-k-cell stage (2.5-hpf), strong SHG
can be observed from centrosomes and mitotic spindles, which are known to be made up of spatially
organized dynamic microtubules and of which the optical centro-symmetry is broken (Campagnola
et al. 2002). On the other hand, based on the sensitivity to optical inhomogeneity, THG contrast can
reflect various interfaces inside a single cell (Yelin and Silberberg 1999; Chu et al. 2001; Schins et al.
2002) such as nuclear membranes, cell membranes, and also the cytoplasmic organelles (Hsieh et al.
2008). Therefore, the cell structures in an embryo and the distribution of the cytoplasmic organelles can
be revealed by THG modality. By using the combined SHG/THG microscope, the dynamic changes of
spindle and membrane between two daughter cells can be imaged in vivo without any exogenous mark-
ers (Figure 14.7). At the initial prophase stage, two centrosomes can be revealed through SHG signals
at the opposite sites of the nucleus, while a circular cell nuclear membrane is clearly visualized through
THG signals (Figure 14.7a). At the prometaphase, the microtubules elongating from the centrioles to
form the spindle can be observed through strong SHG signals, and the alignment of chromosomes in
the center of the cell during the metaphase is disclosed by the broadening and flattening of the spindle
fibers (Figure 14.7b). During the anaphase, the separation of spindle fibers and the alternation of the cell
contour are picked up by the SHG and THG modalities, respectively (Figure 14.7c), while at the end of
the mitosis, the telophase, SHG signals vanish as the spindle microtubules disperse into the cells and
exhibit no more crystalline characteristic (Figure 14.7d).