Combined SHG/THG Imaging - Second Harmonic Generation Imaging - page 337

TABLE 14.2 THG-Revealed and SHG-Revealed Diagnostic Characteristics of Three Types of Skin Diseases,
Including the Compound Nevus, Superficial Spreading Melanoma, and Pigmented Basal Cell Carcinoma

Harmonic Generation Microscopy

Histopathology
Evidences THG SHG

Diagnostic Characteristics

Compound nevus Honeycomb patterns of keratinocytes

Normal keratinization process
Increased number of melanocytes in epidermis
Individually distributed NMs at DEJ
Clustered NMs in dermis
Increased number of fibroblasts
Accumulating melanin in clustered NMs and fibroblasts

Thickening of the
collagen fiber bundles

Supericial
spreading
melanoma
(SSM)

Clearly-revealed superficially spreading MCs with stronger THG contrast
(without staining)
Disrupted honeycomb patterns in epidermis
Scoring different levels of invasions
Distinguishing normal keratinocytes from MCs
Sparsely distributed THG-bright spots in epidermis (higher contents of
melanin)

Collagenous structures
in papillary dermis can
be observed in mildly
invaded regions
(lighter brownish
region)

Pigmented basal
cell carcinoma
(BCC)

Elongating and spindle-like keratinocytes
Higher cell density in epidermis (anomalous keratinization process)
Tumor nodules in dermis
Parallel arrangement of tumor cells at the edge of the nodules
(palisading)
Sparsely THG-bright spots in both epidermis and tumor nodules
(accumulated melanin or melanophages)

Much wavier collagen
fibers at DEJ
Normal collagenous
structures are replaced
by tumor nodules
Collagen fibers enclose
the tumor nodules

Throughout a ~300 μm imaging depth, the in vivo THG imaging is shown to retain submicron lateral
resolution and have less signal degradation than in fixed human skin. Without any staining, the com-
bined SHG/THG microscopy is an excellent tool for in vivo optical skin biopsy and it can provide lots of
significant information for skin disease diagnosis. In the following subsections, the in vivo SHG/THG
imaging of Asian human skin will be introduced, including studies of the in vivo SHG/THG results,
damage evaluation, and spatial resolution.

14.3.5.1 In Vivo SHG/tHG imaging of Asian Human Skin
In the in vivo studies of Asian human skin, investigations were focused on the forearm skin of healthy
Asian volunteers and SHG/THG microscope with backward-collection geometry was applied (Chen et al.
2009a, 2010). Compared with the ex vivo SHG/THG results of the normal human skin shown above,
similar morphological and collagenous information can be observed from the in vivo investigation. As
the in vivo SHG/THG biopsy images shown in Figure 14.19, in epidermis, stratum corneum (Figure
14.19a) is shown not to contain nuclei and have a much stronger THG contrast than other layers of the
epidermis. This strong THG contrast is mainly contributed by the multilayer structure of the stratum
corneum (Tsang 1995; Muller et al. 1998) and the lipid within the corneocytes (Debarre et al. 2006).
Based on the THG contrasts in the cytoplasmic organelles, the cytoplasm of cells in the stratum granulo-
sum (Figure 14.19b), stratum spinosum (Figure 14.19c), and stratum basale (Figure 14.19d) appear THG-
bright, while the nuclei of the cells appear THG-dark. Making use of the contrast between the cytoplasm
and nuclei, the cellular morphology of the epidermis can be easily revealed and the progressive changes
of the nuclear size, cell size, and cell density can all be clearly revealed to show the normal keratinization
process in the epidermis. In the stratum basale, which is rich in melanin contents, the cell borders can be
defined much easier due to much stronger THG signals in the cytoplasm of the basal cells (arrowheads
in Figure 14.19d) and the THG-darker intercellular spaces between neighboring basal cells, where the