Biomedical Engineering Reference
In-Depth Information
properties. Furthermore, some parameters such as mean SHG signal depend upon objective numerical
aperture, detector sensitivity, laser parameters, and other external factors [1-3]. For such instrument-
dependent parameters, absolute values are relatively meaningless without proper instrument calibra-
tion, and information is best gleaned by analysis of trends or by taking a ratio to effectively normalize
the parameter, with the ratio being independent of instrument parameters. For example, careful cali-
bration and optical setup allow for the measurement of forward-to-backward signal ratios [23,25,27].
11.3 SHG imaging of Acellular collagen Gels
11.3.1 introduction
Acellular type I collagen gels are ideal constructs to study the relationship of collagen fiber and network
structural characteristics to SHG signal. These gels are pure and polymerization conditions may be var-
ied independently of collagen concentration to control the aspects of fiber and network microstructure.
At low concentrations (<~9 mg/mL), the fiber network is sparse enough that individual fiber features
can be resolved as well as network features. SHG image parameters correlate with fiber and network
structural features, with implications for using SHG images to estimate bulk mechanical properties.
11.3.2 Quantification of collagen Fiber Shape from Second-Harmonic
images
Altering the polymerization, pH, and temperature of collagen hydrogels significantly influences the
gel microstructure and mechanical properties at a given collagen concentration. In the experiments
described below, gel collagen concentration was kept constant at 4 mg/mL and polymerization was var-
ied between 4°C and 37°C, or between pH 5.5 and 8.5. It was found that increasing pH or temperature
tends to result in longer, thinner collagen fibers, a reduced pore area fraction and size, and an increased
pore density. These characteristics are visible in both SHG and TPF images (Figures 11.1a through 11.1d,
11.1i through 11.1l) and scanning electron microscopy (SEM) images (Figures 11.1e through 11.1h, 11.1m
through 11.1p) of acellular collagen gels. SHG signal to noise is larger than that of TPF signal, a differ-
ence that is likely reflective of the quadratic SHG versus linear TPF concentration dependence, and also
on the generally weak autofluorescence signal from poorly cross-linked collagen. Therefore, incoherent
and homogeneous emission allows the fiber cross-sections to be clearly seen by TPF signal, whereas the
coherent nature of SHG disallows signal generation from dipoles within collagen oriented parallel to the
laser propagation. SEM images have a higher intrinsic resolution and reveal that collagen fibers are actu-
ally closely packed bundles of fibrils, with especially large bundles containing many fibrils at the lower
temperature and pH polymerization conditions. Fiber diameter varies across the polymerization condi-
tions because of differences in the number of fibrils per fiber rather than large changes in fibril diameter.
The measurements of fiber diameter from SHG and SEM images reveal a linear correlation
(Figure 11.2), although the diameters measured from SEM images tend to be smaller (due to dehydration
of the fibrils during sample preparation). Small diameter fibers are visible in SHG (Figures 11.1a, 11.1b,
11.1i, and 11.1j) and SEM images (Figures 11.1e, 11.1f, 11.1m, and 11.1n) of gels polymerized at the lower
temperature and pH values, sometimes independent of larger diameter fibers and sometimes emanating
from the splayed ends of large diameter fibers. However, SHG and SEM images show that large diameter
fibers dominate the space-filling characteristics of these gels. With increasing polymerization tempera-
ture and pH, the hydrogels display a finer and more homogeneous network of fibers.
11.3.3 effects of collagen Fiber Size on Second-Harmonic Signal
In acellular collagen hydrogels, in which collagen is the only significant scattering component,
backward-detected SHG signal primarily results from scattering of forward-generated second-har-
monic photons and from backward-generated SHG from small fibrils (diameters ~10% of λ
2ω
). he
