Lasers in Surgery and Medicine 40:688–694 (2008)

Mechanical Tissue Optical Clearing Devices: Enhancement of Light Penetration in Ex Vivo Porcine Skin and Adipose Tissue Christopher G. Rylander, PhD,1* Thomas E. Milner, PhD,2{ Stepan A. Baranov, PhD,2{ and J. Stuart Nelson, MD, PhD3§ 1 Mechanical Engineering, Biomedical Engineering and Sciences, Virginia Tech, 1880 Pratt Drive, CRC RB XV, Suite 2000, MC 0493, Blacksburg, Virginia 24061 2 Biomedical Engineering, University of Texas at Austin, University Station, C0800, Austin, Texas 78712 3 Beckman Laser Institute and Medical Clinic, University of California Irvine, 1002 Health Sciences Road East, Irvine, California 92612-1475

Background and Objective: The complex morphological structure of tissue and associated variations in the indices of refraction of components therein, provides a highly scattering medium for visible and near-infrared wavelengths of light. Tissue optical clearing permits delivery of light deeper into tissue, potentially improving the capabilities of various light-based therapeutic techniques, such as adipose tissue removal or reshaping. Study Design/Materials and Methods: We report results of a study to evaluate effectiveness of novel mechanical tissue optical clearing devices (TOCD) using white light photography and infrared imaging radiometry (IIR). The TOCD consists of a pin array and vacuum pressure source applied directly to the skin surface. IIR images recorded light absorption and temperature increase of ex vivo porcine skin and adipose during laser irradiation (980 and 1,210 nm) before and after TOCD application. Results: White light photographic images of in vivo human skin demonstrated localized compression and altered visual appearance, indicative of water and blood movement in skin. White light photographic images also showed increased visible light transport through regions of ex vivo porcine skin compressed by TOCD pins. Rate of heating in sub-dermal adipose regions beneath TOCD pins was twofold higher following TOCD application. Conclusions: Results of our study suggest that mechanical optical clearing may provide a means to deliver increased light fluence to dermal and adipose tissues. Lasers Surg. Med. 40:688–694, 2008. ß 2008 Wiley-Liss, Inc. Key words: vacuum; compression; fat; cellulite; reshaping; water transport; refractive index; scattering; absorption; thickness; radiometry; dermis INTRODUCTION Tissue optics, which includes light-based therapeutic applications and diagnostic techniques, has recently received considerable attention. Until recently, optical properties of biological tissues were considered fixed. ß 2008 Wiley-Liss, Inc.

Engineered tissue optics is a new research area that allows reversibly altering light scattering and absorption within naturally turbid tissues in a precise and controlled manner. ‘‘Tissue optical clearing’’ permits delivery of near-collimated light deeper into tissue, potentially improving the capabilities of various optical diagnostic and therapeutic techniques such as new light-based therapies for reshaping or removing adipose tissue. Numerous technical publications describe methods, applications, and potential mechanisms of tissue optical clearing using chemical agents [1–11]. Three hypothesized mechanisms of light scattering reduction induced by chemical agents have been proposed: (1) water transport away from targeted tissue constituents; (2) replacement of interstitial or intracellular water with a chemical agent that better matches the higher refractive index (n) of native proteinaceous structures; and (3) structural modification or dissociation of collagen fibers. Several investigators have suggested that water transport alone can reduce light scattering in soft tissue [12–16]. Water removed from the space between collagen fibrils increases protein and sugar concentrations, decreases refractive index mismatch, and reduces scattering. Because water transport and refractive index matching by exogenous chemical agents can follow sequentially, some investigators do not explicitly distinguish the two mechanisms. The first two mechanisms were initially described by Tuchin et al. [2] while the third was first reported and investigated by Yeh et al. [6]. These three, { Professor. {

Research Fellow. Professor of Surgery and Biomedical Engineering. Contract grant sponsor: NSF; Contract grant numbers: BES9986296, BES0529340; Contract grant sponsor: Candela Corporation; Contract grant sponsor: National Institutes of Health; Contract grant number: AR47551. *Correspondence to: Christopher G. Rylander, PhD, Assistant Professor, Mechanical Engineering, Biomedical Engineering and Sciences, Virginia Tech, 1880 Pratt Drive, CRC RB XV, Suite 2000, MC 0493, Blacksburg, VA 24061. E-mail: [email protected] Accepted 8 September 2008 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/lsm.20718 §

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and possibly other unspecified dynamic mechanisms, may be working synergistically or antagonistically with differing contributions dependent on tissue type and vitality, chemical agent, and delivery method. Because water transport is an important candidate mechanism of optical clearing, we hypothesize that other non-chemical techniques for water redistribution, such as applied mechanical force, should also reduce light scattering in tissue. To test this hypothesis we designed two tissue optical clearing devices (TOCD) each consisting of an array of pins which induce spatially localized tissue compression when connected to a 750 mm Hg vacuum pressure source. We hypothesize that application of vacuum pressure will compress the skin against the TOCD thereby displacing water in tissue underlying the pins permitting increased light fluence in dermal and adipose tissues. Several potential advantages are recognized for optical clearing using the TOCD compared to chemical methods. Mechanical compression using a TOCD is less invasive and presumably safer since no chemical agents are introduced into the tissue and the barrier function of the stratum corneum and epidermis is maintained. TOCD may provide faster onset, more controllable and repeatable optical clearing in comparison to chemical techniques. Additional benefits of mechanical clearing include a means for tissue alignment and registration with a radiant source, and an analgesic effect produced by stimulating or inhibiting sensory nerves in skin by application of mechanical force. MATERIALS AND METHODS Tissue Optical Clearing Device (TOCD) Prototypes Inasmuch as a primary objective of this study was to investigate the effect of application of localized mechanical force on skin optical properties, two TOCD prototypes were designed, constructed and utilized in the experiments described below. The purpose of using two TOCD prototypes was to allow testing of specific experimental techniques to evaluate mechanical optical clearing. The first prototype was designed for photographic imaging of in vivo human skin during TOCD application and infrared imaging radiometry (IIR) of ex vivo porcine skin during 980 nm laser irradiation. The second prototype was designed to focus 1,210 nm laser irradiation at the dermal–adipose junction of ex vivo porcine skin specimens while IIR temperature measurements were recorded. The first TOCD prototype consisted of a monolithic array of pins, a circumscribing brim, and a vacuum pressure source (Fig. 1a). The pin array, which comprised the inner surface of a chamber, was composed of a translucent photoresin which transduced mechanical force to skin. Pin parameters included diameter (1 mm), length (4 mm), lattice geometry (square), packing density (20% fill factor), and tip geometry (hemispherical or flat). The TOCD brim interfaced with the skin surface and formed an airtight seal when vacuum pressure (
750 mm Hg) was applied to the TOCD. The vacuum pressure exerted a mechanical transduction force on the skin, causing stretching and compression of tissue between and underneath the pins, respectively.

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Fig. 1. Simplified cross-sectional schematic diagram of the first (a) and second (b) TOCD prototypes applied to the skin surface.

The second TOCD prototype consisted of an array of sixty-eight 3 mm diameter ball lenses arranged in a hexagonal lattice with a 50% fill factor (Fig. 1b). Ball lenses in this prototype permitted spatial and angular control (focusing) of radiant energy incident onto the skin. Optical radiation was incident on a single ball lens by an optical fiber. Each ball lens was epoxy-bonded to a spherically recessed cavity in a polycarbonate plate. A glass window spacer separated the fiber tip and the polycarbonate plate and allowed full illumination of the ball lens aperture while minimizing internal reflections. This simple optical system consisting of an optical fiber, glass spacer, polycarbonate base, and ball lens was designed to deliver increased light fluence at a depth of 3 mm below the skin surface in dermal and adipose tissues. Tissue Specimens In vivo human skin was chosen for nondestructive experimentation due to its clinical relevance. In vivo volar forearm skin of a Caucasian male volunteer was utilized for photographic experiments. Ex vivo porcine skin specimens were chosen for laser-heating experiments because of potential for pain and/or damage in living tissue and since the dermal thickness and optical properties of this animal model are comparable to those of human skin [17,18]. Porcine skin specimens, consisting of a dermal layer approximately 3 mm thick and an underlying layer of adipose tissue approximately 1 mm thick, were obtained from a local abattoir and stored at 58C until the experiments were performed. White Light Photographic Observations The first TOCD prototype was applied for 30 seconds to the lower volar forearm of a human volunteer using vacuum pressure. White light photographic images of the epidermal surface were recorded through the TOCD during application and after device removal using an Olympus C-3040 digital camera (Tokyo, Japan) with crossed polarizers to eliminate specular reflections from the skin surface. The prototype was also applied with vacuum to porcine skin epidermis for thirty seconds. After TOCD removal locally increased light transport through the skin was

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observed when the specimens were trans-illuminated using a broadband continuous incoherent white light source. Digital photographic images were recorded in the same manner as described above. Infrared Imaging Radiometry—First TOCD Prototype Light transport through ex vivo porcine skin was evaluated using infrared imaging radiometry (IIR) whereby radiometric temperature images were recorded with a Phoenix infrared camera (Goleta, CA) sensitive in the 3–5 mm spectral range (InSb 320256 focal plane array). The prototype was applied directly onto the dermal side of ex vivo porcine skin specimens for 30 seconds. Light emitted from a 980 nm diode laser was delivered through the TOCD into the dermis. The laser was equipped with a scanning mechanism, allowing the 2 mm diameter beam to scan a 6 mm2 area. The infrared camera was positioned on the epidermal side of the tissue specimen to allow direct thermal imaging of light absorption and temperature increase by epidermal melanin. Radiometric temperature images were acquired at a sampling rate of 20 Hz over 10 seconds. Infrared Imaging Radiometry—Second TOCD Prototype The thermal response of subdermal adipose tissue in ex vivo porcine skin specimens due to laser heating was measured with IIR before and after mechanical compression of the prototype against the epidermal surface. Each porcine skin specimen (n ¼ 5) was positioned above the upward-facing mechanical transducers (i.e., ball lenses) to represent a minimal-contact-force control IIR measurement. When vacuum was applied to the TOCD, the epidermal side of the porcine skin specimen moved vertically downward and was compressed into the ball lenses. To induce heating of adipose tissue on the subdermal surface of porcine skin specimens, consideration was given to wavelength of laser irradiation and design of optical focusing elements of TOCD. Optical absorption coefficient of lipids is approximately twice that of water at 1,210 nm [19]; therefore, this wavelength was used to heat selectively adipose tissue while minimizing nonspecific skin heating. Light emitted from a diode laser (1,210 nm) was coupled into a low hydroxide content multimode optical fiber (105 mm core diameter) attached to the fiberoptic connector of the second TOCD prototype. Ball lenses on the TOCD focused incident light (surface irradiance 2.8–3.5 W/cm2) 3 mm deep into porcine skin specimens approximately at the dermal/adipose tissue junction. To maintain steady state operation of the laser with constant radiant output, radiant emission from the diode was switched on/off with a mechanical shutter. Each sample was irradiated for 5 seconds. Acquisition of infrared images by the InSb infrared detector array was triggered by a Ge photoreceiver.

Each porcine skin specimen was irradiated twice while lightly contacting the TOCD without vacuum application (two controls) and then a third time with applied vacuum having started 30 seconds previously and continuing during irradiation. Purpose of the second control irradiation was to test if re-irradiation alone (without vacuum) had any effect on reheating. Subdermal temperature in the porcine skin specimens was maintained in the 20–408C range during 5 seconds irradiation times to avoid thermal denaturation and dehydration of skin specimens. Specimens were allowed to cool to room temperature for 2 minutes between laser irradiations. This protocol allowed computation of the ratio of temperature derivatives (dTratio) corresponding to with- and without-TOCD application for the same skin specimen at the same spatial location. To analyze recorded radiometric images, we selected an area around the pixel with maximum radiant temperature increase. Dimensions of the selected area were 20 pixels20 pixels corresponding to 4 mm2. For this area, we selected the 100 hottest pixels and computed the mean radiometric temperature in the region irradiated by the laser. We recorded 250 infrared radiometric images over a 5 second time period and calculated the corresponding 250 values of radiometric temperatures, T(n) (where n is the frame index between 1 and 250). For radiometric temperature analysis, we calculated the radiometric temperature difference (dT—Eq. 1) between successive time points and the ratio of differential radiometric temperature increase (dTratio—Eq. 2) with and without TOCD vacuum application dT ¼ Tðn þ 1Þ  TðnÞ

ð1Þ

where T(n) is the average radiometric temperature in the extracted region (below the pin) at frame n and T(nþ1) is the average radiometric temperature at the next frame (nþ1) dTratio ¼

dTTOCD dTcontrol

ð2Þ

where dTTOCD is the radiometric temperature difference of the subdermal porcine skin with TOCD vacuum application and dTcontrol is the radiometric temperature difference at the same tissue site without vacuum application. Following this approach, we compared the rates of heating of the subdermal porcine adipose surface directly below a pin with and without the application of a vacuum. For example, the inequality dTratio > 1 indicated that the rate of heating of the subdermal porcine adipose surface is greater when TOCD with vacuum is applied than when the vacuum is not applied.

RESULTS White Light Photographic Observations Application of the first TOCD prototype to human skin resulted in skin compression beneath the pins and skin stretching between the pins. Skin reddening surrounding

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the pins was evident and may be due to displacement of blood from regions directly underneath the pins into surrounding tissue (Fig. 2a). Upon removal of the TOCD, regions of skin compressed by pins appeared darker, indicative of reduced light backscatter (Fig. 2b). Within several minutes following removal of the TOCD redness of skin surrounding pins decreased and redness of compressed skin regions increased. Minor discoloration of skin persisted for approximately 1 day and may have been due to blood leakage from damaged capillaries in compressed skin regions. Thickness of compressed skin regions rebounded to normal within several minutes following TOCD removal. Porcine specimens significantly attenuated visible light transmission. Following application of the first TOCD prototype to the epidermal surface for thirty seconds, both epidermal (Fig. 2c) and dermal (Fig. 2d) views of the porcine skin specimen indicate increased light transmission through mechanically compressed skin regions. Infrared Imaging Radiometry—First TOCD Prototype To indicate pin positions in infrared images of porcine skin, a registration image was initially recorded of the TOCD prototype. TOCD pins were visible in the registration image due to variations in pin emissivity compared to the background. The registration image was superposed on an IIR image of porcine skin recorded after 10 seconds of laser irradiation. Regions of increased epidermal heating in response to 980 nm laser irradiation spatially correlate with TOCD pin positions (Fig. 3). The white box marks the

Fig. 3. IIR measurements of the epidermal side of a porcine skin specimen irradiated (980 nm) on the dermal side through the first TOCD prototype applied to the dermis. Regions of elevated radiometric temperature correspond to TOCD pin positions in the area exposed to laser radiation (white box).

area scanned by the laser beam. Despite absorption of 980 nm light by the polymer base and pins of the TOCD prototype, more light was delivered to regions of epidermis underneath the pins. The flat surface of the pin tip in the first TOCD prototype prevented optical focusing of light into the porcine skin. Infrared Imaging Radiometry—Second TOCD Prototype Plot of radiometric temperature of the subdermal surface vs. irradiation time (Fig. 4a) indicated the subdermal adipose tissue temperature increase was substantially greater when the vacuum was applied as compared to the absence of vacuum. Ratio of differential temperatures (dTratio) with and without TOCD vacuum application (Fig. 4b) remained relatively constant (near 2X) for all five porcine skin specimens. Since no significant difference was observed between heating rates during first and second irradiations (both without vacuum), reheating had no observable effect on the tissue optical properties. DISCUSSION Compression, Water Transport, and Modified Optical Properties

Fig. 2. Photographs of in vivo human skin during application (a) and after removal (b) of the first TOCD prototype. Epidermal (c) and dermal (d) images of ex vivo porcine skin specimen trans-illuminated with visible light subsequent to application of the prototype to the epidermal surface.

A natural gradient of water content as a function of depth exists throughout skin. At the stratum corneum, the outermost layer of epidermis, water content depends on atmospheric humidity and may be as low as 15%. Water content increases with depth approaching the epidermal/ dermal junction reaching a 70% value at 20–75 mm. Water, having a relatively high concentration and permeability, may be displaced from underneath the TOCD pins after vacuum application. We hypothesize that compressive

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heat capacity (c). We assume negligible change in tissue thermal or optical properties at the surface of porcine skin specimens where thermal images were recorded (Figs. 3 and 4, tissue depth
3–4 mm). Inasmuch as the ratio, ma/rc, is not expected to change in the tissue observed using infrared imaging radiometry, the measured increase in rate of heating using the TOCD (Figs. 3 and 4) appears to be primarily due to an increase in local fluence (F). Equation (3) does not account for any heat transfer (conduction or convection) and only considers the temperature increase due to the heat source term. Local fluence in tissue may be approximated by: FðzÞ ¼ F0 emeff z

ð4Þ

where F0 is the irradiance on the surface, z is depth of light penetration into tissue, and meff is the effective attenuation coefficient: qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi meff ¼ 3ma ðma þ m0s Þ ð5Þ

Fig. 4. a: Radiometric temperature T(n) with and without vacuum application to second TOCD prototype. b: Ratio of differential temperature increase (dTratio) corresponding to curves in (a).

stress in the tissue underneath TOCD pins forces water laterally to lower stress tissue regions surrounding the pins. Supporting evidence includes increased volume and reddening of skin surrounding pins during TOCD application as indicated in Figure 2. Compressive stress is a common driving force for both water and blood transport; therefore, observed movement of blood, although through the vesicular pathway, likely indicates direction of water movement. In light-based therapeutics, successful treatment outcome may depend on a desired temperature increase in selected tissue regions resulting in destruction of targeted chromophores or regions, while maintaining temperature below the damage threshold in non-targeted tissue regions. For example, potential laser-based adipose tissue reshaping or removal procedures would benefit from selective heating of subdermal adipose tissue while minimizing heating in overlying skin. Rate of temperature increase dT(r,z)/dt in tissue at position (r,z) due to light absorption is given by, dTðr; zÞ ma F ¼ dt rc

ð3Þ

and is dependent on local tissue absorption coefficient (ma), local optical fluence (F), tissue density (r), and specific

which depends on the absorption (ma) and reduced scattering coefficients (m0s ) of overlying tissue. TOCD application causes a significant reduction in tissue thickness measured from the surface to a physiological location (z0) as demonstrated in Figure 2. Because of the exponential dependence of fluence on depth (Eq. 4), decrease in z0 will result in increase in F(z0). This effect is more significant for deeper targeted depths such as adipose tissue. Also, considering the large fraction of free (unbound) water in skin, the potential exists for a large decrease in tissue thickness due to TOCD compression. Variability of water content in skin also affects ma and m0s at infrared wavelengths. Dependence of optical absorption coefficient on water content in skin has been described by Brugmans et al. [20]: ma ¼ Wma;water þ ð1  WÞma;0

ð6Þ

where W is the unitless water mass fraction, ma,water is the optical absorption coefficient of water (¼1.25 cm1 for l ¼ 1,210 nm), and ma,0 is the water independent (fully dehydrated) absorption coefficient in skin (¼0.75 cm1 for l ¼ 1,210 nm) [21]. This relationship is graphically presented in Figure 5 and illustrates the significant dependence of absorption coefficient on skin water content at this wavelength. Application of a TOCD may displace water greatest in superficial skin regions just under pins, with diminishing affect at increasing depth; therefore, absorption coefficient at 1,210 nm is expected to increase with increase depth underneath pins. Skin acts as a highly scattering medium for optical wavelengths due to its complex and nonhomogeneous morphological structure. Light scattering in biological tissues is caused primarily by variations in electronic polarizability at optical frequencies, which may be characterized by variations in the optical index of refraction, n. Tissue constituents such as collagen (70% of dry weight of dermis), lipids, water, cells and their organelles all have slightly different indices of refraction. Water, with its lower index of refraction contributes significantly to the optical

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Fig. 5. Dependence of skin optical absorption coefficient (1,210 nm) on tissue water mass fraction.

index of refraction mismatch, Dn, giving rise to light scattering. Scattering coefficient may be locally modified by: (1) increased collagen fiber density; and (2) increased proteoglycan concentration in ground substance causing greater intrinsic refractive index matching [11–16]. Water transport out of selected skin regions may reduce Dn and resulting light scattering, reducing fluence in more superficial tissue layers, and thus increasing fluence in targeted deeper tissue layers such as adipose tissue. Potential TOCD Use Light attenuation in native tissue due to scattering and absorption constrains the useful penetration depth for imaging and therapeutic applications. Optical clearing is well suited to increase the light fluence in deep tissue structures such as adipose. For other applications, however, optical clearing may be non-beneficial or possibly detrimental. Epidermal resurfacing, for example, commonly utilizes 2,940 nm wavelength Er:YAG laser to ablate the top 20 mm of water-absorbing epidermal tissue while sparing deeper cutaneous tissue. Delivery of this wavelength through TOCD pins into compressed epidermis could reduce superficial ablation and therapeutic outcome for epidermal resurfacing. Tissue optical clearing is an assistive technology that may aid in achieving a more desirable adipose treatment outcome and may be used in conjunction with other technologies such as lipid-selective photothermolysis and dynamic cooling of skin. TOCD may be implemented as a single or multi-use accessory that can attach to a conventional laser handpiece. Different TOCD configurations may be optimized to treat specific areas of tissue, and the clinician may choose to utilize several different TOCD configurations during a single patient treatment. A TOCD can be designed to interface with light delivery, vacuum, and cooling components within the laser handpiece. Vacuum pressure within the TOCD causes tissue stretching between pins and compression beneath pins. Total force exerted over the area of tissue between pins of

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the second TOCD prototype was 45 N, which was balanced by the reaction force of compression beneath pins. In a clinical setting, the operator may impose additional forces on treatment sites by pushing or pulling the TOCD relative to the patient’s body. Pushing the TOCD against the tissue will increase compressive forces under pins and aid optical clearing, whereas pulling the TOCD away from the tissue will result in the opposite effect. If the TOCD is implemented clinically, operators may be trained to impose a minimal compressive force against the patient’s skin. All experiments performed in this study utilized only the forces generated by the vacuum pump. We are presently uncertain whether TOCD-assisted laser procedures will provide advantages for the treatment of cellulite and fat reduction. Successful treatment outcome depends on many factors including the time–temperature history of the tissue, volume of treated tissue, and the wound healing process. TOCD treatment may modify all of these factors compared to non-TOCD treatment. TOCD treatment may increase the depth of damage in localized regions of adipose. Non-targeted regions (between TOCD pins) may be spared from damage and may beneficially aid in the remodeling process of the adjacent damaged tissue. If a more aggressive treatment is desired the TOCD procedure can be repeated multiple times at slightly offset positions such that tissue untreated on the first pass is treated during a subsequent pass. In this manner total treatment volume can be increased at the cost of increased treatment time. Additional studies are necessary to determine how TOCD-assisted laser procedures can provide advantages for the treatment of cellulite and fat reduction. CONCLUSIONS Other researchers have investigated pressure effects on tissue optical properties and measured increased transmission, decreased reflectance, and decreased sample thickness [15,22]. Using an inverse-adding-doubling method, these researchers reported increased absorption and scattering coefficients with increasing compression pressure. An important distinction exists, however, between these and our experimental methods. In previous studies, a spring-loaded compression apparatus delivered uniform pressure distribution across the entire tissue specimen. The TOCD prototypes used in our experiments induced spatially localized compression zones underneath the pins, permitting water movement laterally between the pins. Results of our study suggest that our TOCD prototypes laterally displace interstitial water and blood underneath the pins, inducing zones of dehydration, reducing tissue thickness, and possibly modifying optical properties. Tissue compression may improve radiant throughput by reducing the physical pathlength to a physiological target in the tissue. Absorption coefficient may be reduced by displacement of light-absorbing chromophores such as water and blood. Scattering coefficient may be reduced by intrinsic refractive index matching and increased scatterer

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density. Reduced tissue thickness and modified optical properties may permit increased fluence at targeted chromophores. More accurate measurement of changes in thickness, absorption, and scattering as a function of tissue depth are required for better understanding contributions of each to increased fluence and rate of heating.

ACKNOWLEDGMENTS This research was funded in part by NSF (Grant No. BES9986296 and No. BES0529340), and a research grant from Candela Corporation. JSN was supported by the National Institutes of Health (AR47551). The mechanical optical clearing methodology described in this manuscript is contained within U.S. Patent Application Serial No. 11/ 502,687, ‘‘Systems, devices, and methods for optically clearing tissue,’’ filed August 11, 2006, awarded to Thomas E. Milner, J. Stuart Nelson, Christopher G. Rylander, and Oliver F. Stumpp, and assigned to The University of Texas and Regents of the University of California. Licensing rights to the patent have been obtained by DermaLucent LLC. The authors (CGR, TEM and JSN) disclose financial interest in DermaLucent LLC. The authors would like to thank Oliver Stumpp for his help with the photographic observations and IIR using the first TDCD prototype.

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