Biomedical Engineering Reference
In-Depth Information
12
SHG and Multiphoton
Fluorescence Imaging
of the Eye
Chiu-Mei Hsueh
National Taiwan University
Po-Sheng Hu
National Cheng-Kung
University
Wen Lo
National Taiwan University
Shean-Jen Chen
National Cheng-Kung
University
12.1 Introduction ......................................................................................271
12.2 Principles of Second Harmonic Generation Microscopy...........273
Second Harmonic Generation Microscopy • Multiphoton
Microscopy Instrumentation
12.3 Visualization of Normal Corneal and Surrounding Tissues ..... 274
12.4 Imaging and Characterization of Pathological Corneas ............276
12.5 Future in Clinical Diagnosis ...........................................................283
References......................................................................................................285
Hsin-Yuan Tan
National Taiwan University
Chang Gung University
Chen-Yuan Dong
National Taiwan University
12.1 introduction
As the organ responsible for vision, the eye plays a vital role in our communication with the external
world. Among the intricate components that constitute the eye, collagen-rich cornea is the key optical
element responsible for most of the eye's refractive properties. Although the cornea only represents
one-sixth of the outer coating of the eye, its dome-shaped and optically transparent structure contrib-
utes to more than two-thirds of the eye's focusing power. In addition to its collagen content, corneal
epithelium is maintained by stem cells located at limbus, which constitutes the peripheral boundary of
the cornea. Beyond the limbus, the sclera forms the remaining ocular surface and is responsible for
structural integrity and protection of intraocular contents. Like the cornea, the sclera is covered by the
epithelial conjunctiva and is mainly composed of collagen fibers. Nonetheless, it is the unique alignment
of the collagen fibers that is responsible for corneal transparency [1,2].
The unique transparency and important visual function of the cornea is of intensive interest to
researchers. Structurally, the cornea can be divided into the following five layers: epithelium, Bowman's
membrane, stroma, Descemet's membrane, and the endothelium [3]. The epithelium is composed of five
to six thin layers of squamous, nonkeratinized epithelial cells. Below the epithelium exists a randomly
and densely arranged collagen fibrous layer (type I, III, V, and VI) known as Bowman's membrane,
which separates the epithelium from the stroma. The stroma, which makes up 90% of the cornea, con-
sists of orthogonally stacked lamella layers. Each lamella layer is composed of long, parallel-aligned
collagen fibrils, whose main composition is type I. Interspersed within the collagen lamella are the kera-
tocytes whose main functions are maintaining corneal transparency, synthesizing cellular components,
and promoting wound healing. The Descemet's membrane connected to the posterior stroma is an acel-
lular and homogeneous layer rich in basement membrane glycoproteins, laminin, and type IV collagen.
The deepest layer of the cornea is the endothelium composed of a single squamous cell layer. In oph-
thalmology, physiological studies at the organ level have depended heavily on histological techniques
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