Abstract. Biomedical optics and photomedicine applications are challenged by the turbidity of most biological tissue systems. Nonreactive, biocompatible chemical agents can induce a reversible reduction in optical scattering of collagenous tissues such as human skin. Herein we show that a chemical agent's tissue optical clearing potential is directly related to its collagen solubility, providing a rational design basis for effective, percutaneous formulations. © Success of light-based therapies depends on sufficient photon energy reaching subsurface targets. This can be problematic since most biological tissues are highly light scattering, dispersing much of the incident photon energy from intended targets, thereby reducing therapeutic efficacy. For dermatologic applications, the optical properties of human skin, 1 in particular light scattering by ubiquitous, high-order collagen structures within the dermis, 2 prevent delivery of collimated light to the desired targets. These same properties prove to be formidable barriers to the collection of light ͑or optical signal͒ from within as in the case of diagnostic fluorescence or whole-body bioluminescence studies. As a consequence, many optical devices have been designed for use in the nearinfrared spectral region where tissue light scattering and absorption are minimal.Traditionally, tissue optical properties have been considered fixed. However, after tissue immersion in particular nonreactive chemical agents, a temporary reduction in light scattering has been demonstrated.3 Optical clearing can be dramatic as tissue becomes visibly transparent and loses mechanical compliance. Upon immersion in physiological saline, tissue turbidity and mechanical properties return to their native states. 4 Common properties of these chemical agents such as sugars and sugar-alcohols have suggested refractive index matching with native tissue components, in particular collagen, and dehydration are possible mechanisms of tissue optical clearing.3 However, a mechanistic understanding of tissue optical clearing using refractive index matching remains to be substantiated. Previous studies suggest a consequence and necessary condition of tissue optical clearing is the destabilization of highorder collagen structures; after tissue immersion in compounds used for fixation, optical clearing does not occur. 5 In fact, many of the chemical agents used for optical clearing also have been used in collagen self-assembly studies to characterize and understand the dynamics of fibrillogenesis. 6Herein we use solubility tests to characterize chemical agentcollagen interaction for a series of sugar-alcohols ͑ethylene glycol, glycerol, xylitol, and sorbitol͒ and propanediols ͑1,2-propanediol and 1,3-propanediol͒ and correlate this data with optical clearing potential measurements in rodent and human skin in vitro.We first characterized the inhibitory effects of chemical agents on collagen self-assembly. In vitro self-assembly of solubilized ͑pHϳ 3͒ rodent tail collagen I ͑BD Biosciences͒ into fibri...

Reduction of optical scattering in turbid biological tissues using nonreactive chemical agents has potential applications for light-based diagnostics and therapeutics. Optical clearing effects by exogenous chemical agents, in particular sugars and sugar alcohols, have been found to be temporary with tissue rehydration. Applications with dermatologic laser therapies are now being investigated, but suffer from the inability of studied agents to penetrate the superficial layers of human skin. Selection, design, and refinement of topically effective chemical agents are hindered by a lack of fundamental understanding of tissue clearing mechanisms. We present recent work, particularly from the biochemistry community, detailing molecular interactions between chemical agents and collagen. This body of work demonstrates the perturbative effects of sugars and sugar alcohols on collagen high-order structures at micro- and nanometer length scales by screening noncovalent bonding forces. In addition, these studies emphasize the nonreactive nature of agent-collagen interactions and the ability of noncovalent bonding forces to recover with agent removal and drive reassembly of destabilized collagen structures. A mechanism of tissue optical clearing is proposed based on agent destabilization of high-order collagen structures.

Background and Objective: Light scattering from collagen within skin limits light-based therapeutics while increasing the risk of epidermal thermal injury. Specific chemicals show the ability to reduce light scattering by reversibly altering the optical properties of skin. This study examines the correlation between collagen solubility and the optical clearing potential (OCP) of sugars and sugar-alcohols using in vitro rodent skin. Materials and Methods: Collagen solubility in dextrose, fructose, sucrose, and sorbitol was measured using near-UV spectroscopy. Light transmittance, reflectance, and rodent skin thickness were measured (giving skin reduced scattering coefficient) before and after exposure of the dermal surface to sugars and sugar-alcohols. OCP was calculated as the ratio of reduced scattering coefficients before and after exposures. Results: Dextrose, fructose, sucrose, and sorbitol had at least twice the collagen solubility and twice the OCP as compared to glycerol. In general, collagen solubility correlated with each agent's ability to optically clear rodent skin.Conclusion: These results demonstrate that sugar and sugar-alcohol interaction with collagen are a primary event in tissue optical clearing.

Abstract. Molecular interactions of optical clearing agents were investigated using a combination of molecular dynamics ͑MD͒ simulations and optical spectroscopy. For a series of sugar alcohols with low to high optical clearing potential, Raman spectroscopy and integrating sphere measurements were used to quantitatively characterize tissue water loss and reduction in light scattering following agent exposures. The rate of tissue water loss was found to correlate with agent optical clearing potential, but equivalent tissue optical clearing was measured in native and fixed tissue in vitro, given long-enough exposure times to the polyol series. MD simulations showed that the rate of tissue optical clearing correlated with the preferential formation of hydrogen bond bridges between agent and collagen. Hydrogen bond bridge formation disrupts the collagen hydration layer and facilitates replacement by a chemical agent to homogenize tissue refractive index. However, the reduction in tissue light scattering did not correlate with the agent index of refraction. Our results suggest that a necessary property of optical clearing agents is hyperosmolarity to tissue, but that the most effective agents with the highest rates of optical clearing are a subset with the highest collagen solubilities.

Functional optical characterization of disease progression and response to therapy suffers from loss of spatial resolution and imaging depth due to scattering. Here we report on the ability of dimethyl sulfoxide (DMSO) alone to reduce the optical scattering of skin. We observed a threefold reduction in the scattering of skin with topical DMSO application. With an in vivo window chamber model, we observed a threefold increase in light transmittance through the preparation and enhanced visualization of subsurface microvasculature. Collectively, our data demonstrate the potential of DMSO alone to mitigate effects of scattering, which we expect will improve molecular imaging studies.