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J. Photochem. Photobiol. B: Biol., 16 (1992) 127-140

Optical properties of in vitro epidermis and their possible relationship with optical properties of in vivo skin Renato

Marchesini”,+,

Claudio

.Clementeb,

Emanuele

Pignoli”

and Marco

Brambilla’

“Department of Health Physics and bDepariment of Pathology and Cytopathology Istituto Nazionale per lo Studio e la Cura dei Tumori, via Venezinn I, I-20133 Milan0 (Italy) ‘Department of Health Physics, Ospedale Ma&ore, C. so Mazzini 18, Novara (Italy) (Received

March 23, 1992; accepted

July 24, 1992)

Abstract Using a spectrophotometer with an internal integrating sphere, the absorption (CL,) and reduced scattering (CL,‘) coefficients of in vitro epidermis were evaluated from reflectance and transmittance measurements. pa and Pi’ varied from 24 to 0.2 cm-r, and from 32 to 21 cm-’ respectively, on passing from 400 to 800 nm. Moreover, using an external integrating sphere, the reflectance spectrum of in vivo skin was compared with the reflectance spectrum calculated with a Monte Carlo model, in which the mean values of pa and IL,’ and different anisotropy parameters were used as input data. In vivo results show that the principle of similarity is entirely valid for wavelengths greater than 600 nm and may be considered a good approximation in the 400-600 nm band, and suggest that optical characteristics of in vivo skin may be inferred from reflectance measurements.

Keywords: Epidermis, transmittance, Monte

absorption coefficient, Carlo model.

scattering

coefficient,

reflectance,

1. Introduction The efficacy of therapeutic and diagnostic procedures that use laser sources and involve skin disorders often depends on the knowledge of the optical characteristics of skin since they determine the propagation and fluence distribution of optical radiation in tissue. Basically, the description of light propagation in optically thick media is provided by the radiative transfer theory [l]. This deals with the transport of energy within a medium and ignores the wave nature of light. Solutions of the radiative transfer equation can be obtained with different analytical or numerical methods, each of which involves simplified physical assumptions and knowledge of the optical properties of the irradiated tissue. Detailed reviews on the models of radiation transport and on the influence of optical properties of tissue on fluence distribution have recently been published by Patterson et al. [2, 31. Few studies have measured the optical characteristics of different human tissues over a wide spectral range [4, 51. In particular, the epidermis has received little ‘Author

to whom correspondence

loll-1344/92/$5.00

should be addressed.

0 1992 - Elsevier Sequoia. All rights reserved

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attention even though this tissue is the first of the cellular layers involved in skin-light interaction. The aim of the present study was twofold: firstly, to determine, in the wavelength range 400-800 nm, the optical characteristics of in vitro epidermis; secondly, to evaluate whether reflectance measurements of in vivo skin, with the aid of the evaluated in vitro parameters, can give useful information about the optical characteristics of in vivo skin. In the framework of the diffusion approximation [2], the absorption coefficient pa and the reduced scattering coefficient ps’ =~(l -g) of in vitro epidermis were determined by indirect methods that employ measurements of reflectance and transmittance. Furthermore, to assess the accuracy of the calculated parameters independently of the theoretical assumptions inherent in the diffusion approximation, we applied a Monte Carlo model to evaluate reflectance and transmittance by using as input data the previously calculated optical characteristics. Finally, utilizing the mean values of pa and b’ and selected values of the anisotropy parameter g, we used *a Monte Carlo model to calculate the reflectance spectrum of an optically very thick sample. The resultant curve was compared with the experimental reflectance spectrum obtained from measurements in vivo by using an integrating sphere. Comparison of the results showed that: (i) the principle of similarity may be applied in modelling light transport in skin; and (ii) in vivo reflectance measurements may be used as an indirect method to estimate the optical parameters of skin.

2. Materials

and methods

2.1. Instrumentation The main components of the instrumentation were as follows. (1) A double beam UV/vis spectrophotometer (Model Lambda 5, Perkin-Elmer, Germany). (2) An internal integrating sphere 7.6 cm in diameter (Perkin-Elmer) used for the in vitro measurements of reflectance and transmittance. The sphere is provided with two entrance (i.e. for the reference and sample beams) and two exit ports (9x 17 mm”). The throughput is greater than 0.95. (3) An external integrating sphere 12 cm in diameter (PerkinElmer) used for the in vivo measurements of reflectance. The external sphere was connected to the spectrophotometer by two flexible fiber optic bundles (i.e. for the reference and sample beams). The sample beam is directed towards the 1 cm diameter sample port, whereas the reference beam is directed towards the inner wall next to the sample port. The throughput of the sphere was between 0.95 and 0.97. Both the spheres, internally coated with barium sulphate, were operated in comparison mode [6] since measurements were performed by contemporaneously having in place both sample and standard. The reflectivity of barium sulphate was assumed to be 1.0 from 400 to 800 nm. Transmittance and/or reflectance measurements were performed between 400 and 800 nm at a scan speed of 120 nm min-‘. Spectral resolution of the incident light beam was 2 nm and 4 nm when using the internal and external integrating spheres, respectively. Reflectance and transmittance spectra were recorded and stored in a personal computer for subsequent data analysis. 2.2. In vitro measurements Ten samples of epidermis were obtained from specimens of normal, full-thickness skin removed from 10 Caucasian patients who underwent oncological surgery. Skin specimens derived from different anatomical locations: upper leg (n = 2); lower back

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(n = 2); breast (n = 2); thigh (n = 2); abdomen (n = 1); and groin (n = 1). Separation of epidermis from dermis was achieved by following the procedure suggested by Bamberger et al. [7]. Briefly, after removal of the subcutaneous fat by scraping, the skin specimen (approximately 1x2 cm2) was placed on a temperature-controlled heating plate with the dermis in contact with the plate. A 1 mm diameter thermocouple was vertically positioned and gently pressed against the epidermis to allow measurement of the temperature reached by the superficial skin layer. After 2 min at 49 “C, the skin sample was removed from the hot plate. Epidermis was carefully separated from dermis and immediately placed over the surface of a saline solution to avoid wrinkles and to moisten the sample. The epidermal layer was directly collected from the saline solution with a metal frame (30x 15 mm’) with a rectangular opening (11 X 8 mm2) and then covered with an identical framework to keep the epidermis in place. The opening covered with epidermis was alternately placed on the front and rear ports of the internal integrating sphere for diffuse transmittance and reflectance measurements respectively. The incident, quasi-collimated light was directed toward the stratum comeum in both cases. The whole procedure was performed within 1 h of the surgical excision. Following spectroscopic measurements, epidermis samples were submitted to histological preparation, and the thickness of each sample was determined under microscopic observation as the mean of 10 readings. 2.3. In vivo measurements Ten reflectance measurements were performed on in vivo skin, in similar anatomical sites as for the in vitro measurements, in 10 Caucasian volunteers. The external integrating sphere was gently positioned in contact with the area under investigation. Reflectance measurement was made on the air-stratum comeum interface, as it was for the in vitro procedure. Room lighting was maintained as dim as possible. 2.4. Evaluation of optical characteristics of epidemis To derive pL, and A’ of epidermis, we employed the 1-D diffusion approximation of the radiative transfer equation originally proposed by Groenhuis et al. [8], assuming a reflectance value of 0.034 due to the air-stratum corneum interface (equivalent to a refractive index of 1.45), and a value of 0.3 for the internal reflectance coefficient [9, lo]. The computer algorithm used to derive the optical characteristics from reflectance and transmittance measurements has been described elsewhere [ll], and the computer source program was kindly provided by Dr. L. I. Grossweiner, Chicago. Briefly, using the experimental values of reflectance and transmittance and employing a fast-fitting algorithm based on Newton’s two-parameter method, the values of pa and A’ = ~(1 -g) were calculated. The uniqueness of the computer fitting has been tested. 2.5. Model of light propagation Experimental measurements of transmittance and/or reflectance were compared with the results obtained by simulating the fate of photons in the light-tissue interaction by means of a previously developed Monte Carlo model [12]. To determine the type of interaction process (i.e., absorption or scattering) and the related free path length, the following procedure was applied. At each interaction site, two free path lengths, dk and d,, were obtained by sampling two random numbers, X, and X,, uniformly distributed in the interval (OJ], so that: dk= -(l/&*lnXk

and d,=

-(l/&*lnX,

where p-L,and pL, have their usual meanings. If d,

Optical properties of in vitro epidermis and their possible relationship with optical properties of in vivo skin.

Using a spectrophotometer with an internal integrating sphere, the absorption (mu a) and reduced scattering (mu s') coefficients of in vitro epidermis...
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