Biomedical Engineering Reference
In-Depth Information
molecules which self-assemble into fibrils (diameter: 10-300 nm, length: up to several hundred μm). In
tissues such as tendons, collagen I fibrils form bundles or fibers with diameters between 0.5 and 3 μm.
Other collagens, such as collagen IV, do not form fibrils, because of numerous interruptions in the heli-
cal sequence by non-collagenous domains. Individual collagen IV molecules assemble to form a two-
dimensional network in the matrix which is found mainly in basement membranes.
The macromolecular organization of collagens is crucial in the organs' architecture. The quan-
tity and distribution of the various types of collagens result from a balance between synthesis and
assembly mechanisms on one hand, and degradation mechanisms on the other, which are regulated
by complex signal pathways. In response to various injuries, the three-dimensional (3D) distribu-
tion of collagen is modified and fibrillar collagen accumulates in the tissue. This cascade of events
is called fibrosis. It alters the structure of affected organs and leads to their functional failure. For
instance, renal fibrosis induced by hypertensive inflammatory and mechanical stress is now the first
cause of renal insufficiency. However, the relationships between tissue injury, enzymes activity, and
tissue remodeling are only poorly understood because of the limitations of conventional imaging
techniques. It is therefore crucial to develop new approaches to study such processes taking place at
different scales.
In that respect, the most important issues are the visualization of the fibrosis
3D architecture
in intact
biological tissues and the determination of
quantitative
indexes of collagen fibrosis. Visualization of the
fibrosis network would give insight into the biological mechanisms of fibrosis progression in relationship
with other components of the tissue. It necessitates a multimodal approach to visualize simultaneously
fibrillar collagens and various proteins of interest or pathological processes, including inflammatory
processes. Determination of fibrosis quantitative indexes would enable unambiguous quantization of
the role of various enzymes that regulate synthesis/assembly and degradation of collagen in the fibrosis
progression and, ultimately, in the fibrosis regression. Such a quantitative imaging method has to be
specific to fibrillar collagen that appears to be a better predictor of severe pathological progression than
nonfibrillar collagen because it is more resistant to proteolysis.
15.1.2 SHG imaging of Fibrillar collagens
In this context, second-harmonic generation (SHG) microscopy appears as a valuable technique since
fibrillar collagens exhibit strong endogenous SHG signals. SHG is a nonlinear optical (or multiphoton)
process that is complementary to two-photon excited fluorescence (2PEF) and that appears at the har-
monic frequency of the laser excitation (that is half the excitation wavelength). Endogenous SHG signals
have been observed in a limited number of biological compounds, mainly collagen, skeletal muscle,
starch, and so on (Campagnola et al. 2002, Zipfel et al. 2003). SHG signal from collagen is the largest
SHG signal in mammals and is specific to fibrillar types of collagen, as verified by comparison with
immunochemical labeling (Zoumi et al. 2002, Brown et al. 2003, Strupler et al. 2007).
The reason why SHG is specific to fibrillar collagen is related to the physics of this nonlinear signal.
The next section will therefore present the physical origin of SHG endogenous signal in collagen. Readers
who are not familiar with nonlinear optics and chemical-physics may just read the summary given as
the last subsection. In the third section, we will review SHG imaging of the lung, kidney, and liver
fibrotic tissues and discuss the contribution of this technique to fibrosis imaging. The fourth section will
be devoted to fibrosis quantization by use of SHG microscopy. Finally, we will discuss the advantages
and limitations of SHG microscopy for fibrosis scoring and give some perspectives.
15.2 Physical origin of SHG Response from collagen
and Specificity to Fibrillar collagens
The physics of collagen SHG must be considered at two different scales. First, we must identify what
chemical entities exhibit a nonlinear response within the collagen triple helix, and second, we have to
