Biomedical Engineering Reference
In-Depth Information
A large SHG response is obtained when the harmonophores are aligned in the same direction so that
the second-harmonic fields add up in a constructive way as depicted in Figure 15.1b. On the contrary,
a centrosymmetric distribution of harmonophores results in destructive interferences and vanishing
SHG signal at the macromolecular scale. The SHG intensity is finally obtained as the square of the total
SHG field and exhibits a quadratic dependence with the number of aligned harmonophores. SHG is
therefore specific to
dense noncentro-symmetric
macromolecular organizations.
Let us now examine how the SHG signal builds up within the collagen triple helix. Since all the pep-
tide bonds are somehow aligned in the same direction, the second-harmonic fields add up mainly in a
constructive way. Moreover, the collagen triple helix is a very dense peptide structure. The SHG signal
is therefore efficiently amplified along the compact and rigid triple helix (see Figure 15.1c). This mecha-
nism is supported by HRS measurements of the nonlinear response of type I collagen and of a short
collagen-like model peptide (Deniset-Besseau et al. 2009). Coherent amplification however saturates
in collagen I whose length (300 nm) is close to the optical wavelength (400 nm) because of phase shifts
along the triple helix. Model calculations of this effect provide an estimation of the collagen nonlinear
response as a function of the length of the triple helical domain, which is the only relevant parameter
in that respect (Deniset-Besseau et al. 2009). Advanced quantum chemistry calculations have also been
performed recently to test this additive model. They qualitatively reproduce experimental measure-
ments although some theoretical refinements are required (Loison and Simon 2010).
This mechanism of coherent amplification also applies at the macromolecular level. Accordingly,
SHG signals have been reported in fibrillar collagens because of the dense and aligned organization of
collagen molecules within collagen fibrils (Campagnola et al. 2002, Zoumi et al. 2002, Cox et al. 2003,
Zipfel et al. 2003, Strupler et al. 2007). On the contrary, nonfibrillar collagen IV does not exhibit any
SHG signal because it is organized as a centrosymmetric network with low density (Strupler et al. 2007).
Aligned collagen IV molecules deposited as a thin film do however exhibit an SHG response (Pena et al.
2005). It proves that the vanishing SHG response from collagen IV in tissues is related to its macromo-
lecular organization, not to the precise amino-acid sequence (Strupler et al. 2007). In that respect, SHG
imaging is very different from immunochemical techniques.
At higher scale, for a set of fibrils (or a fiber), the SHG signal is a complex process because fibrils may
be aligned in opposite directions within the focal volume, so that their radiated harmonic fields cancel
out. This effect was demonstrated in rat-tail tendons by use of piezo response force microscopy (Rivard
et al. 2011). It results in a decreased SHG signal in the forward direction, while backward-directed SHG
signal is less modified because it corresponds to smaller coherence lengths. Nevertheless, forward SHG
signal usually exceeds backward SHG signal because phase matching is favored in the forward direction.
(It is 10 times larger in Achille tendon, for instance (Legare et al. 2007).) Phase matching along the focal
volume is also sensitive to the Gouy phase shift like in third-harmonic microscopy (Barad et al. 1997,
Débarre et al. 2005, Williams et al. 2005, Olivier and Beaurepaire 2008). The SHG signal observed in
collagenous tissues must therefore be considered as a complex interference pattern from all the fibrils in
the focal volume, not as a morphological image of the fibrillar structure (LaComb et al. 2008b, Strupler
and Schanne-Klein 2010, Rivard et al. 2011).
15.2.3 Summary
To sum up, the strong SHG response of fibrillar collagen is not related to the presence of a strong
harmonophore in the amino-acid sequence but to the tight alignment of many weakly efficient har-
monophores that are the peptide bonds. This tight alignment toward the same direction results in
an efficient coherent amplification at all the hierarchical levels of the fibrillar collagen organization:
single α chain => triple helix => fibril => fiber. Fibrils may however be organized in a complex man-
ner within the focal volume and strong focusing, moreover, complicate phase-matching processes.
Consequently, the resulting image is a complex interference process that may not fully reproduce the
morphology of the fibrils while highly specific to fibrillar collagens. Note that an SHG signal can also
