Biomedical Engineering Reference
In-Depth Information
FIgurE 14.1
In vivo
THG images of a zebrafish embryo obtained at different depths beneath the top chorion
surface. The structures of the (a) top chorion, (b) top cellular layer, (c)-(e) yolk cells, (f) bottom cellular layer, and
(g) bottom chorion can be observed within a depth of ~1.5 mm. The corresponding imaging planes are indicated
in (h). Scale bar: 50 μm.
2009), setting the average excitation power of both lasers to 40 mW for imaging of porcine skin, the Ti:S
SHG light almost disappeared beyond a depth of 200 μm, while the Cr:F SHG signals were still detect-
able even at a depth of 350 μm. This result strongly indicates improved penetrability under the Cr:F
excitation. In previous studies of various animal models, a high penetrability (several hundred microns)
can be found in most cases, but this varies widely among different tissues. Especially for transparent tis-
sues, like zebrafish embryos (Chu et al. 2003; Chen et al. 2006) and mouse cornea (Chen et al. 2008), an
extremely high penetrability can be obtained, enabling imaging deep inside the tissues, while still pre-
serving a high spatial resolution. For example, Cr:F-based SHG/THG microscopy (forward-collection
geometry) can be used for
in vivo
imaging of zebrafish embryo with a diameter of 1.5 mm (Chu et al.
2003). The upper chorion structures, top cellular layer, yolk cells, bottom cellular layer, and bottom
chorion structures can all be observed throughout the whole embryo (Figures 14.1a through 14.1g).
The corresponding imaging planes are indicated in Figure 14.1h. Even at an imaging depth of ~1.5 mm
beneath the top chorion surface, the <1 μm diameter granular canals of the bottom chorion surface can
be clearly distinguished, demonstrating the preservation of submicron resolution (500 nm resolution at
a depth of 1.5 mm). Cr:F-based SHG/THG microscopy (backward-collection geometry) has been used
to image an excised mouse eye. It can be seen that based on the transparency of the mouse cornea, a
penetration depth of ~700 μm can be achieved, and the structures of the outermost corneal epithelium,
corneal stroma, corneal endothelium, and even the lens fibers can be observed within the imaging depth
inside the mouse eye (Chen et al. 2009b). In addition to the high penetrability demonstrated in the
zebrafish embryos and mouse cornea, Cr:F-based SHG/THG microscopy has demonstrated a ~300-μm
high penetrability of fixed human skin (Tai et al. 2005).
14.2 cr:F-Based SHG/tHG Microscopy
14.2.1 Principle of SHG and tHG
In contrast to the linear process, SHG and THG are both higher-order nonlinear processes. As implied
by the name “nonlinear optics,” the polarization intensity of a molecule produced in the nonlinear
optical process has a nonlinear dependence on the electric field of the excitation light. The polarization
