Biomedical Engineering Reference
In-Depth Information
polarization. This is the reason why the tendon cleared in 50% glycerol exhibits a completely different trend
of β versus depth, where forward signal anisotropy increases through 110 μm total thickness (doubled
due to swelling), and changes from 0.3 at the top of the sample to 0.85 at the last optical plane at the
bottom (Figure 8.14, circles).
The anisotropy increases with the decrease in the propagation distance to the exit from the sample.
Still, due to residual scattering at both wavelengths, the anisotropy does not reach a value of 1.0 as one
would expect for the highly ordered collagen fibrils in the tendon. Again, similar results have been
found in the muscle, although the anisotropy values are inherently lower.
Overall, polarization dependencies are largely preserved in both the tendon and striated muscle
cleared with 50% glycerol and consistent with the reduction of scattering that we measured by integrat-
ing spheres [6].
8.10 Final Remarks
The potential safety issues with the use of clearing agents in vivo must be thoroughly investigated, includ-
ing considerations for the OCA concentrations and exposure times. Optical clearing causes swelling in
the striated muscle and tendon, although there is no significant disruption of tissue structure, and the
process is reversible in the case of tendon and 50% glycerol. Still, there remains a concern over possible
alteration of higher-order protein structures by OCA's. At the same time, shrinking has been observed in
glycerol-treated gastrointestinal tissue due to dehydration of the intercellular space [8]. Additionally, all
the ex vivo samples can be easily cleaned with a uniformly distributed agent over a few hours of incuba-
tion. When used in vivo , both topically or injected into the tissue, the clearing agent will be subject to
further diffusion from the region of interest. Hence, even if toxicity is not a problem, in the clinic, we will
not have the luxury of exposure for several hours for complete replacement of interstitial water with an
OCA; hence, there is no guarantee of uniform reduction of scattering. Even if optical clearing is limited to
ex vivo tissues, it still offers advantages over traditional histological staining methods and complements
them with subresolution information through a much less time- and labor-consuming approach.
Regarding SHG specifically, the F / B ratio of SHG intensity is highly dependent on the extent of opti-
cal clearing, due to the effects of both scattered SHG component and of directly emitted backward SHG
signal. Fibril separation is also increased compared to that of the native tissue. Most likely the F / B metric
will not be consistent for diagnostic purposes, although visualization deeper into the tissue has been
shown to improve. On the contrary, SHG polarization measurements greatly benefit from optical clear-
ing, since polarization signatures are randomized only in two to three scattering events, but are retained
through at least 100-200 μm of cleared tissue, as demonstrated in the muscle and tendon. Finally, the
need remains to fully understand the mechanism of optical clearing during complex interactions of
OCAs and tissues to make the process predictable and controllable.
References
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SPIE, Bellubgham, WA, USA.
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Clement. 2001. Increased extracellular matrix remodeling is associated with tumor progression in
human hepatocellular carcinomas. Hepatology 34:82-88.
3. Nadiarnykh, O., R. B. Lacomb, M. A. Brewer, and P. J. Campagnola. 2010. Alterations of the extracel-
lular matrix in ovarian cancer studied by second harmonic generation imaging microscopy. BMC
Cancer 10:94.
4. Plotnikov, S. V., A. Kenny, S. Walsh, B. Zubrowski, C. Joseph, V. L. Scranton, G. A. Kuchel, D. Dauser,
M. Xu, C. Pilbeam, D. Adams, R. Dougherty, P. J. Campagnola, and W. A. Mohler. 2008. Measurement
of muscle disease by quantitative second-harmonic generation imaging. J. Biomed. Opt . 13:044018.
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