Biomedical Engineering Reference
In-Depth Information
(a)
(b)
(c)
2000
2000
2000
1000
1000
1000
pH
Temperature
0
0
0
0
0
10 20
Polymerization
temperature (°C)
30
40
0
6
Polymerization pH
7
8
9
1
2
3
4
d
SHG
FIgurE 11.3
(a) Mean segmented SHG signal versus polymerization temperature of acellular collagen gels.
(b) Mean segmented SHG signal versus polymerization pH of acellular collagen gels. (c) Mean segmented SHG
signal plotted versus average fiber diameter measured from SHG images,
d
SHG
, for acellular gels polymerized at
pH 5.5, 6.5, 7.5, and 8.5, or temperatures 4°C, 14°C, 24°C, and 37°C. The error bars are standard error of the mean.
(Reprinted from
Biophys J
., 92, Raub, C. B. et al., Noninvasive assessment of collagen gel microstructure and
mechanics using multiphoton microscopy, 2212-2222, Copyright 2007;
Biophys J
., 94, Raub, C. B. et al., Image
correlation spectroscopy of multiphoton images correlates with collagen mechanical properties, 2361-2373,
Copyright 2008, with permission from Elsevier.)
dependency of epi-detected SHG signal on collagen fiber size can be seen in Figures 11.3a and b, for
4 mg/mL acellular collagen gels polymerized from 4°C to 37°C and pH 5.5-11, respectively. The SHG
signal has been segmented to exclude void regions, so that the measurements represent average signal
values only from collagen. Plotting the segmented SHG signal versus fiber diameter shows a direct,
linear correlation for both varying pH (
R
2
= 0.96) and temperature (
R
2
= 0.85) polymerization condi-
tions, with an offset attributable to the differing detector gain (Figure 11.3c). The effect of increasing
mean fiber diameter is to increase the scattering cross-section of the fiber as well as bulk scattering
within the tissue, which is thus able to scatter more forward-directed SHG photons into the epi-
configured detectors.
11.3.4 effects of Acellular collagen Gel concentration
on Second-Harmonic Signal
While the concentration dependence of second-harmonic signal scales with the square of generating
dipole concentration, changes in the bulk collagen concentration of acellular gels may increase both
the average density of collagen within signal-containing pixels and the relative volume fraction of
collagen within the gel. The effect of increasing fiber number density can be seen in SHG images for
fine- (Figures 11.4a-c) and coarse-structured gels (Figures 11.4d-f) with low collagen concentrations
(1.5-9 mg/mL). For these collagen gels, SHG signal mean intensity (Figure 11.4g) and area fraction
(Figure 11.4h) increase linearly with collagen content. The linearity of the signal increase is robust to
changes in collagen fiber morphology observable from the SHG images and may be attributed sim-
ply to changes in collagen fiber number density and concomitant decreasing of void volume fraction,
rather than increased fibril packing within pixels, which would introduce a nonlinear (second-order)
dependence of the signal on collagen concentration. The scattering coefficient within collagen gels is
expected to increase by ~2.9 cm
−1
per 1 mg/mL of collagen, from 4.3 cm
−1
at 1.5 mg/mL, and 26 cm
−1
at 9 mg/mL [87], and may contribute to linear increases in SHG signal intensity with collagen con-
centration. A similar linear trend in SHG area fraction versus collagen concentration (Figure 11.4h)
suggests that for these acellular, low-density collagen gels, the microstructure determines SHG image
parameters.
