Determination of chemical agent optical clearing potential using in vitro human skin

Department of Dermatology, University of California, Irvine, California 92612Background and Objectives: Optical clearing is amethod that temporarily reduces the optical scattering ofbiological tissues.Study Design/Materials and Methods: To determinethe optical clearing potential (OCP) of various chemicalagents, we examined the change in reduced scatteringcoefficientsofinvitrohumanskinafterapplicationdirectlyto the dermal side.Results/Conclusions: We found that the mean agentOCP did not correlate with refractive index or osmolality.Lipophilic hyperosmotic agents had a mean OCP less thanunity,indicating increasedoptical scattering. LasersSurg.Med. 36:72–75, 2005. 2005 Wiley-Liss, Inc.Key words: hyperosmotic agents; glycerol; scattering;dermatology; port wine stainsINTRODUCTIONIn biomedical applications, light is used for diagnosticimaging and therapeutics. The penetration depth of light inbiological tissue is restricted by absorption and scatteringevents. At visible and near infrared wavelengths, opticalscatteringdominatesoverabsorptionandismuchmoresigni-ficant in reducing light penetration into biological tissues.Optical clearing is a method for inducing a transient re-duction in optical scattering [1]. Studies have demonstratedtheincreasedpenetrationdepth andcontrastinopticalimag-ing [1–8]. Using a novel chemical agent (CA), Khan et al. [9]havedemonstratedforthefirsttimeinvivoopticalclearingofhuman skin. Various mechanisms for optical clearing havebeen proposed, including refractive index matching [1,5],dehydration [5,10,11], and collagen dissociation [12].Hyperosmotic CAs with refractive indices similar to thatof collagen have been shown to increase tissue transpar-ency. From results obtained with three CAs, Vargas et al.[13] suggested that increased light transmittance is pro-portionaltoCAosmolality.Inthisstudy,wequantitativelycharacterized a select group of hyperosmotic CAs. By mea-suring the optical clearing potential (OCP, defined below),we set out to determine whether there is a correlationbetween changes in optical scattering and osmolality andrefractive index.MATERIALS AND METHODSChemical AgentsWe studied three different groups of CAs: hydroxy-terminated, organic solvents, and organic acids as sum-marized in Table 1. Refractive index was measured with arefractometer (Cole-Parmer, Vernon Hills, IL). Osmolalitywas measured with a freezing point osmometer (AdvancedInstruments, Norwood, MA). Isotonic saline was used as anegative control (i.e.,no optical clearing expected).Glycerol(13 M)was used as a positive control (i.e., optical clearingexpected).Skin SamplesCryopreserved, dermatomed human skin (CommunityTissueServices,Dayton,OH)wasthawedtoroomtempera-ture( 258C).Asingleedgedrazorbladewasusedtocuttheskin into 2.5 cm 2.5 cm samples. Sample thicknessvariedbetween0.4and1mm,asdeterminedbyplacingthesample between two glass slides of known thickness andmeasuring the thickness of the sample/glass combinationwith a micrometer (Mitutoyo, City of Industry, CA). Tominimizeerrorduetosamplecompression,werecordedthesample thickness once the fine adjustment screw on themicrometer clicked once. Measurements on each samplewere initiated within 2 hours after thawing.Franz Diffusion ChamberAfter removal from between the glass slides, each skinsample was mounted in a Franz diffusion cell [14]. Thelowerreservoirwasfilledwithisotonicsaline.Eachsample