Biomedical Engineering Reference
In-Depth Information
The first issue was recently addressed by measuring the second-order hyperpolarizability of the
collagen triple helix by use of hyper Rayleigh experiments (Deniset-Besseau et al. 2009). It was shown to
be
β
= 1.25 × 10
−27
esu for type I collagen from rat tail (relative to the water response: 0.56 × 10
−27
esu).
Moreover, the response for any collagen type was obtained as a function of the length of the triple helical
domain, which was shown to be the only relevant parameter in that issue. For that purpose, we devel-
oped model calculation of the coherent summation of the response of all the peptides' bonds tightly
aligned along the triple helix (Deniset-Besseau et al. 2009).
The second issue is a highly complicated task and requires more experimental data and more theoret-
ical work. The second-order coherent response of a given distribution of collagen molecules may be cal-
culated using similar approaches to the ones developed for membrane dyes (Moreaux et al. 2000), single
fibrils (Williams et al. 2005), or an assembly of fibrils (LaComb et al. 2008b, Strupler and Schanne-Klein
2010). However, the inverse problem cannot be solved unambiguously without complementary data, for
instance measuring the ratio of forward- to backward-SHG signals or varying the collection numerical
aperture (Williams et al. 2005, Chu et al. 2007, LaComb et al. 2008a, Rivard et al. 2011). Polarization-
resolved SHG microscopy may also help to get an insight to the 3D distribution of fibrillar collagen
within the focal volume (Roth and Freund 1981, Stoller et al. 2002, 2003, Williams et al. 2005, Erikson
et al. 2007, Han et al. 2008) provided a careful analysis of polarization distortions in collagenous tissues
(Mansfield et al. 2008, Nadiarnykh and Campagnola 2009, Aït-Belkacem et al. 2010, Gusachenko et al.
2010).
15.5.2.2 improvement of image Analysis
SHG quantization of fibrosis extent is based on filtering and thresholding algorithms. These algorithms
may be improved to better retrieve significant signals. For instance, Otsu threshold segmentation
and erosion and dilation removal of grainy noise were used to quantify liver fibrosis (Sun et al. 2008).
Other algorithms have been proposed in the context of collagen quantization in engineered tissues. For
instance, adaptative thresholding procedure followed by zeroing of non-interconnected pixels was used
to quantify the density of collagen matrices (Bayan et al. 2009). Complementary information may also
be obtained by processing the images to obtain orientation indexes of the collagen fibrils as developed
for engineered tissues or cornea (Raub et al. 2008, Bayan et al. 2009, Matteini et al. 2009, Bowles et al.
2010). 2D Fourier transform or Hough transform algorithms were used to map the average orientation
and the disorder (entropy) of the collagen fibrillar network. Texture analysis was also proposed in car-
tilage SHG images (Werkmeister et al. 2010).
However, all these approaches are restricted to sequential 2D image processing and subsequent aver-
aging on z-stacks to obtain volume indexes. It would be of great interest to develop direct 3D image pro-
cessing to better retrieve the 3D fibrillar distribution. For that purpose, a 3D morphological analysis was
recently proposed and successfully discriminated different fibrillar organizations in collagen matrices
(Altendorf and Jeulin 2009, Altendorf et al. 2012). Similar approaches should be developed for describ-
ing the millimeter-scale architecture of fibrosis. For instance, kidney fibrosis in murine models was
shown to exhibit a radial interconnected distribution through the cortical region with striking continu-
ity between perivascular, periglomerular, and peritubular fibrosis (Strupler et al. 2008). Quantitative
analysis of this distribution would be of great interest for monitoring fibrosis progression and should
benefit from 3D morphological approaches.
15.5.2.3 Applicability for
In Vivo
Diagnosis
Application of SHG microscopy to
in vivo
imaging and fibrosis diagnosis would be very interesting.
There are two issues in that respect. First, one has to develop fibered SHG microscopes in order to better
access the fibrotic organs. It requires complex developments, and work is currently under progress to
achieve such advanced endoscopes. The second issue is regarding the penetration depth of SHG imag-
ing. Typical penetration depth in highly dense and scattering tissues such as kidney, liver, or skin is
a few hundreds of micrometers. It could be further improved using adaptative optics (Jesacher et al.
