Biomedical Engineering Reference
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be observed in
aligned liquid
solutions of collagen as reported in collagen liquid crystalline organiza-
tions (Deniset-Besseau et al. 2010). While these concentrated solutions at acidic pH do not form fibrils,
they show aligned molecular domains with sufficient density to exhibit a significant SHG signal.
Altogether, SHG microscopy is a structural probe of the macromolecular organization of collagen
and it is highly specific to fibrillar collagens in tissues. In that respect, it appears to be a relevant method
for fibrosis imaging.
15.3 Second-Harmonic imaging of tissue Fibrosis
15.3.1 Multiphoton imaging of Lung, Kidney, and Liver Fibrosis
SHG potential for fibrosis assessment was first mentioned in 2003 in a paper by Cox et al. (2003). SHG
imaging and scoring of fibrosis has then been implemented by a few groups and mainly applied to lung,
renal, and liver fibrosis (Banavar et al. 2006, Pena et al. 2007, Strupler et al. 2007, 2008, Sun et al. 2008,
2010, Tai et al. 2009b, Gailhouste et al. 2010, He et al. 2010, Raub et al. 2010). It proved relevant to visu-
alize specifically collagen fibrosis in unstained tissues both in animal models and in human biopsies.
SHG microscopy has been usually combined with 2PEF microscopy to take advantage of intrinsic fluo-
rescence from various tissue components to visualize the tissue morphology.
Figure 15.2 displays such combined SHG/2PEF images of an intact fibrotic lung from a bleomycin-
treated mouse. The SHG signal reveals the accumulation of fibrillar collagen in heterogeneously dis-
tributed areas in the alveolar interstitium, which is characteristic of bleomycin-induced fibrosis and of
idiopathic pulmonary fibrosis in humans (Pena et al. 2007). Another example of multiphoton imaging
of fibrosis is displayed in Figure 15.3, which shows coronal slices of murine control and fibrotic kidneys.
In the cortical region, SHG reveals collagen fibrils in the Bowman's capsule and in the tubular inter-
stitium. SHG signal is also observed from fibrillar collagen in the adventitia of arcuate arteries and of
arterioles in the interstitium. The 3D distribution of fibrillar collagen in the arterial adventitia and in the
tubular interstitium is better visualized in 3D reconstructions obtained from image stacks acquired at
increasing depths within the thick tissue (see Figure 15.3d) (Strupler et al. 2008). Finally, SHG images of
rat liver fibrosis are displayed in Figure 15.4, which shows morphological changes of collagen distribu-
tion during fibrosis: collagen deposition radiates around the portal tract and then extends to form septa
that connect and achieve complete bridges with adjacent portal tract and central veins (Sun et al. 2008).
The above examples refer to fibrotic samples from animal models that were mostly used to develop opti-
mized protocols for fibrosis imaging (Strupler et al. 2007, Sun et al. 2008). Nevertheless, human fibrotic
tissues have also been imaged by use of SHG microscopy as shown in Figures 15.5 through 15.7. Figure
15.5 displays SHG/2PEF images of normal and fibrotic human lung histological sections: SHG image of
idiopathic pulmonary fibrosis highlights marked architectural changes with fibrosis onset and distorted
alveolar spaces (Figure 15.5b). It reveals the distribution of collagen mainly in the periphery of fibroblas-
tic foci, with fibroblasts and thinner fibrillar components in the center (Figure 15.5c) (Pena et al. 2007).
Human biopsies of fibrotic kidney implants show similar collagen distribution as in murine samples with
accumulation of fibrillar collagen in the Bowman's capsule and in the tubular interstitium (see Figure
15.6) (Strupler et al. 2008). The distribution of collagen fibrosis in human liver biopsies is also similar to the
one observed in rat tissues with deposits of fibrillar septa connecting portal tracts, although the collagen
distribution may differ according to the underlying liver disease (see Figure 15.7) (Gailhouste et al. 2010).
15.3.2 Advantages of SHG Microscopy
The studies reported in the last section highlight many advantages of SHG microscopy compared to
conventional techniques for fibrosis assessment. The main advantage is obviously the high specificity of
SHG microscopy to fibrillar collagens, as explained in the previous section. The other advantages that
often stem from the latter one are detailed in the following.
