Biomedical Engineering Reference
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cells. In addition to the SHG-revealed collagenous information, more cellular, morphological, and even
molecular information can be provided by THG modality. Since THG is well known to be sensitive to
local optical inhomogeneity (Squier et al. 1998; Peleg et al. 1999; Yelin and Silberberg 1999) and lipid
(Debarre et al., 2006), the outermost stratum corneum, composed of multilayers of lipid and corneocytes,
can be revealed by THG modality with ultrastrong THG contrast (arrows in Figures 14.12a and 14.12b).
Beneath the stratum corneum, the honeycomb architectures of the stratum granulosum (arrowheads
in Figure 14.12a), the stratum spinosum (Figure 14.12b), and the stratum basale (Figure 14.12c) can be
revealed by THG modality based on THG contrasts arising from the cytoplasmic organelles (Hsieh et al.
2008). As shown in the THG images, the cytoplasm of the keratinocytes appears THG-bright, while the
nuclei of the keratinocytes appear THG-dark. In contrast to the strong THG intensity in the stratum
corneum, the THG intensity in the keratinocytes is shown to be much weaker, but can still provide
clear cellular morphological information of the epidermis. At the dermo-epidermis junction (Figure
14.12c), the collagen structures of the dermal papillae are shown by SHG modality. Surrounding the
SHG-revealed dermal papillae, THG reflects the basal cells with a circular locus, and the size of the locus
progressively increases as the imaging plan moves deeper (Figures 14.12d and 14.12e). Splitting the dif-
ferent channels of Figure 14.12e into separate THG (Figure 14.12e-T) and SHG (Figure 14.12e-S) images,
abundant THG signals can be found to arise from the elastic fibers (arrows in Figure 14.12e-T) and the
fibroblasts collagen fiber boundaries, which are shown in the regions overlapping the simultaneous SHG
signals. Figure 14.12i shows the axial histology section of the mouse back skin, while the axial SHG/
THG image is shown in Figure 14.12j. In the axial SHG/THG image, the layer structures of the skin,
including the THG-brighter stratum corneum (arrow in Figures 14.12i and 14.12j), epidermis (E), and
dermis (D), are shown with a high axial resolution and have strong correspondences to the histology
image. Moreover, the average thickness of the epidermis measured from the SHG/THG image is found
to coincide with that measured from the histology section; this consistency indicates the capability of
THG modality for investigating the pathological changes of the epidermis thickness.
Although the basic morphological structures and chemical composition of mouse skin are similar in
different parts of mouse body, slight differences can be found among the mouse ear, back, and abdomen
skin. Generally, the ear skin is a little thinner than the back skin and the fat tissues are more abundant in
the abdomen skin. Owing to the regional variations, the imaging capability of SHG/THG microscopy may
vary in different parts of mouse skin, and the application scope may thus be broadened. Figures 14.13a
through 14.13f show a series of SHG/THG images of the mouse ear skin obtained at different depths beneath
the skin surface. In the ear skin and back skin, only a slight difference exists in the thickness of epidermis.
However, in the ear skin, the sebaceous glands around the hair follicles appear at a more superficial layer of
the dermis and a higher density of the sebaceous glands can be found in contrast to the back skin. Since the
lipid-filled cells in the sebaceous glands progressively accumulate lipids until the plasma membrane breaks
down, the strong THG contrast in the sebaceous glands can be mostly contributed by the lipid (Debarre
et al. 2006). As shown in Figures 14.13e and 14.13f, the sebaceous glands can extend to the deeper dermis
with a >60 μm total length in axial. In skin, it is believed that there are discrete populations of epidermal
stem cells in the stratum basale, hair follicles, sebaceous glands, apocrine glands, and eccrine glands. To
study the activation of these stem cells, an imaging tool with the ability for
in vivo
observation is desired.
Our results of the mouse skin not only show that THG microscopy has strong imaging contrast in the hair
follicles and the sebaceous glands, but also indicate the capability of THG microscopy for studies of the
epidermal stem cells. On the other hand, in the mouse abdomen skin, where more abundant fat cells can be
found, the polygonal fat cells are revealed by THG modality at about 70 μm (Figure 14.13g), while the loose
collagen fibers surrounding the fat cells are revealed by SHG modality. Even at 120 μm (Figure 14.13h), the
fat cells can still be shown with a submicron spatial resolution.
14.3.3.2 Mouse eye
According to the histological results of the cornea (Maurice 1984; Ramaesh et al. 2004), the cornea can
be roughly divided into three main components—the corneal epithelium (EP), corneal stroma (CS),
