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Biomedical Optics & Medical Imaging

Advances in optical clearing of skin in vivo

A novel, noninvasive, controllable technique is suitable for high-resolution observation of subcutaneous structures.
7 April 2011, SPIE Newsroom. DOI: 10.1117/2.1201103.003567

Optical-imaging technology has become one of the most promising approaches in biomedicine, since it can be used to deduce the structure and function of tissues in vivo. However, one limitation to its application is skin cover, which prevents light access to subcutaneous targets. Tissue optical clearing1 can improve the penetration depth of light by immersing tissues in optical-clearing agents (OCAs), such as glycerol and glucose, with high refractive indices and dermal permeability. Recently, neural circuits of drosophila mushroom bodies were mapped in high-resolution 3D detail under the microscope using OCAs.2 Such a technique would be useful for subcutaneous observation, although whether OCAs used in vitro are effective and safe for use in vivo has yet to be determined. To address these issues, our research has focused on mechanisms of optical clearing in vivo and biocompatibility of OCAs. Our aim is to develop a noninvasive, controllable optical-clearing technique for opening a ‘transparent window’ through skin to determine subcutaneous microvascular structures and functions at high resolution.

We first explored the physical mechanisms of optical clearing—using tissue phantoms—by mixing the liquid phantom with OCAs. Both experimental results and theoretical predictions demonstrate that higher OCA refractive indices improve optical-clearing efficacy as long as OCAs do not change the structure of the sample.3 However, when treating biological tissues, OCAs may alter the sample structure, thus complicating the optical-clearing mechanisms. Using rat dorsal skin as model, we injected OCAs at various concentrations. We found that while increasing the OCA concentration improved optical clearing, there was a noticeable concentration-dependent decrease in dermal thickness (see Figure 1). Most likely caused by dehydration of the dermis, this is advantageous because it improves refractive-index matching and thins the major scattering layer.4 Additionally, increased OCA concentrations resulted in decreased collagen-fiber diameter and increased fiber regularity, which play an important role in the mechanism for OCA-induced optical clearing in skin.4 These results differ from in vitro immersion experiments of skin, where collagen-fiber dissociation or fracture occurred.5

Figure 1. Images of dermal collagen fibers taken by transmission-electron microscopy (TEM) and second-harmonic generation (SHG) 10min after injection of optical-clearing agents (OCAs). (a) Blank control, (b) 20, (c) 30, and (d) 75% by volume glycerol.

While dermal injection allows optical clearing, we were interested in developing noninvasive techniques. Before topical OCAs are used in vivo, we need to evaluate possible effects on tissue function. We used laser-speckle-contrast imaging to observe changes in chick chorioallantoic membranes (CAMs) after application of varying OCA concentrations.6 We investigated both short- and long-term effects by monitoring changes of blood flow and development of blood vessels in CAMs, respectively. We found that OCAs reduced the local blood-flow velocity and constricted, or blocked, blood vessels. While blood flow recovered to varying degrees, this depended on the OCA type and concentration. The extent to which new blood vessels developed also depended on OCA type and concentration (see Figure 2). Observing vasculature in this way provides a new method for determining OCA biocompatibility. Additionally, OCAs are less effective when used topically than when injected subcutaneously. The outermost layer of skin—the stratum corneum (SC)—blocks the majority of the OCA and, thus, diminishes the optical-clearing effect. To reduce the SC-barrier effect, we used a newly developed penetration enhancer, thiazone: benzisothiazol-3(2H)-one-2-butyl-1,1-dioxide. Topical application of a mixture of thiazone and OCA can improve visualization of subcutaneous microvessels.7 We used laser-speckle temporal-contrast analysis to visualize dermal blood flow at high resolution, which was not previously possible using OCAs alone. Post-imaging treatment with saline allowed skin recovery (see Figure 3).

Figure 2. Photographs (grayscale) and speckle velocity maps (color) of blood vessels in chick chorioallantoic membranes before, 31.5min after, and 48h after topical application of different concentrations of OCA: (a) 100, (b) 50, and (c) 25% by volume glycerol, (d) 40 and (e) 20% by weight glucose, and (f) physiological saline.

Figure 3. Rat dorsal skin at a range of times after topical application of polyethylene glycol 400 (optical clearing agent) mixed with thiazone (penetration enhancer). (a) Dorsal photographs, (b) pane-region photographs, (c) pane-region temporal-contrast maps.

In summary, we have demonstrated that optical clearing is a promising tool for in vivo, noninvasive, and controllable subcutaneous imaging. Combined with optical-imaging technology, such as fluorescence and laser-speckle-contrast imaging, we achieved improved image contrast and resolution. In future, our optical-clearing methods could be used for optical diagnosis of peripheral blood-vessel disease. In particular, the technique will be useful for investigating mechanisms of tumorigenesis and progression as well as evaluating the effectiveness of tumor treatment. These important applications to clinical medicine constitute our ongoing work.

This work was supported by the National Natural Science Foundation of China (grants 30770552 and 30911120074). We thank Valery V. Tuchin of Saratov State University (Russia) for his help.

Dan Zhu, Xiang Wen, Jing Wang
Britton Chance Center for Biomedical Photonics
Wuhan National Laboratory for Optoelectronics
Huazhong University of Science and Technology
Wuhan, China

Dan Zhu is a professor. She has authored more than 80 papers in the field of tissue optical theory and techniques. Her current research interests are in optical clearing of skin in vivo.

Xiang Wen is a PhD candidate in biomedical engeering.

Jing Wang is a PhD candidate in biomedical engeering.

1. V. V. Tuchin, Optical Clearing of Tissues and Blood, SPIE Press, 2006.
2. H.-H. Lin, J. S.-Y. Lai, A.-L. Chin, Y.-C. Chen, A.-S. Chiang, A map of olfactory representation in the drosophila mushroom body, Cell 128, pp. 1205-1217, 2007. doi:10.1016/j.cell.2007.03.006
3. X. Wen, V. V. Tuchin, Q. Luo, D. Zhu, Controling the scattering of intralipid by using optical clearing agents, Phys. Med. Biol. 54, pp. 6917-6930, 2009. doi:10.1088/0031-9155/54/22/011
4. X. Wen, Z. Mao, Z. Han, V. V. Tuchin, D. Zhu, In vivo skin optical clearing by glycerol solutions: mechanism, J. Biophoton. 3, pp. 44-52, 2010. doi:10.1002/jbio.201090004
5. J. Hirshburg, B. Choi, J. S. Nelson, A. T. Yeh, Collagen solubility correlates with skin optical clearing, J. Biomed. Opt. 11, pp. 040501, 2006. doi:10.1117/1.2220527
6. D. Zhu, J. Zhang, H. Cui, Z. Mao, P. Li, Q. Luo, Short-term and long-term effects of optical clearing agents on blood vessels in chick chorioallantoic membrane, J. Biomed. Opt. 13, pp. 021106, 2008. doi:10.1117/1.2907169
7. D. Zhu, J. Wang, Z. Zhi, X. Wen, Q. Luo, Imaging dermal blood flow through the intact rat skin with an optical clearing method, J. Biomed. Opt. 15, pp. 026008, 2010. doi:10.1117/1.3369739