Biomedical Engineering Reference
In-Depth Information
a micron. Each of the α-chains (~300 nm long) within the triple helix is a polypeptide chain of high
regularity, composed of repetitive -Gly-X-Y- sequences, where X and Y can be any amino acid, though
X is commonly proline and Y hydroxyproline. This degree of order resembles that of a nonlinear crystal
with periodically arrayed unicells. SHG has been shown to be sensitive to both the fibril and fiber levels
of organization [38]. A broad range of collagen tissues have so far successfully imaged with SHG tech-
niques, which includes self-assembled collagen gels [40], the epimyseum and perimyseum wrapping of
muscle [6], bone [41], fish scale [6], skin [42], rat or mice tendon [43-45], cornea [46-49], and ovary [38].
We will discuss the unique application of SHG imaging for collagen tissues in more detail below with
two examples, explicitly showing the structural sensitivity.
Cornea, the important organ of the visual system for organisms, is composed essentially of all type
I collagen [50], and it has been studied intensely using SHG imaging [47-49]. Han et al. found that
cornea collagen fibers generate strong SHG signal, and the emission almost completely follows the
direction of the incident laser, consistent with the transparency of cornea. At the same time, SHG
signal generated in the closely related sclera tissue (also largely composed of collagen fibers) is more
backward directed than forward (Figure 4.8) [47]. Scanning electron microscopy indicates that cor-
neal collagen fibrils have a very uniform diameter of ~25 nm, and are regularly packed into layers
of extended structures (lamellae) resembling crystalline lattices much larger than λ
SHG
. In contrast,
scleral fibrils possess diameters that are widely distributed in the range of 25-300 nm, and present a
random morphology of smaller structures [50]. Clearly, microstructures of the collagen tissues indi-
cate that cornea resembles a crystal more than sclera does, and the general relaxed phase-matching
conditions described earlier would predict predominantly harmonic generation in the forward direc-
tion, as is experimentally observed. In contrast, the smaller structures of sclera lead to a mixture of
forward and backward components.
FIgurE 4.8
SHG imaging of collagen fibrils in forward and backward directions of cornea (a, b) and sclera
(c, d). Bars: 10 μm. (Reproduced from Han, M., G. Giese, and J. F. Bille. 2005.
Opt. Express
13:5791-5797. With
permission.)
