Lasers in Surgery and Medicine 37:308–313 (2005) Relationship Between Damaged Fraction and Reflected Spectra of Denaturing Tissues Hiroyuki Yoshimura, PhD, 1 * John A. Viator, PhD, 2 and Steven L. Jacques, PhD 3 1 Faculty of Engineering, Chiba University, Chiba, Japan 2 Department of Biological Engineering, University of Missouri, Columbia, Missouri, 65211 3 Department of Dermatology, Oregon Health & Science University, Portland, Oregon, 97239 Background and Objectives: During thermal therapy of tissue, such as induced by microwave heating, the initiation of denaturation should be monitored for proper thermal dosage. Additionally, denaturation should be confined to the pathologic volume, while preserving surrounding healthy tissue. The relationship between the damaged fraction and reflected spectra of denaturing tissues was investigated for a variation of the temperature of the tissues. Study Design/Materials and Methods: Denaturation of muscle, liver, and milk was studied in vitro by measuring the temperature-varying reflectance spectrum as heating occurs. A high-resolution fiber optic spectrometer was used to measure the reflectance changes. Temperature was monitored using a thermocouple embedded within the tissue along the side of the optical fiber probe. Results: The values of average free energy to initiate denaturation in muscle and liver at about 608C were 94.8 and 96.3 kJ/mole, respectively. The reflectance spectra increased in amplitude for muscle and liver, and the peak shifted from approximately 700 to 720 nm in accordance with the damage fraction of tissue. The reflectance spectrum for milk was essentially unchanged. Conclusions: Spectral changes from heated muscle and liver reflect denaturation of proteins contained therein. The spectral information at 800 nm can be used to de- termine the average free energy for the initiation of denaturation. Lasers Surg. Med. 37:308 – 313, 2005. ß 2005 Wiley-Liss, Inc. Key words: biological tissue; denaturation; free energy; optical fiber; temperature; thermal therapy INTRODUCTION In thermal therapy, such as hyperthermia and coagula- tion therapy, temperature control is important to achieve high-quality treatment of diseased tissue. The therapeutic temperatures in hyperthermia and coagulation therapy are between 42 and 458C and above 608C, respectively. In some studies [1–3], a fiberoptic thermometer has been used to measure temperature during thermal therapy due to its immunity from electromagnetic interference during micro- wave irradiation of tissue. These probes are expensive, thus we propose an alternative to monitoring tissue denatura- tion using spectral measurements. Several studies on thermal damage of biological tissue based on the Arrhenius type thermal damage integral have been conducted since the pioneering work of denaturation of porcine skin by Henriques [4]. For example, Meijerink et al. experimentally investigated the thermal damage of egg-albumen (egg-white) [5]. London numerically simu- lated a benign prostate hyperplasia (BPH) diode laser treatment system [6]. Gerstman et al. experimentally in- vestigated the laser induced thermal damage to the retina [7]. Pfefer et al. experimentally investigated the thermal damage of whole blood induced by pulsed laser [8]. Recently, several studies on the relationship between the thermal damage of liver tissue and the optical spectroscopy have also been conducted in vitro [9] and in vivo [10,11]. As a result, it was shown that fluorescence and diffuse reflec- tance spectroscopy have the potential to monitor thermal damage of liver tissue in thermal therapy. Lin et al. have shown that the behavior of normalized fluorescence and diffuse reflectance spectra of liver tissue by heating was compared to the degree of thermal damage based on the Arrhenius type thermal damage integral [9]. The degree of thermal damage, however, was numerically calculated under the assumption that the temperature of the tissue is constant during the heating, and therefore, was not related directly to the temperature of the tissue. In other studies, the histological evaluations were only used to assess the thermal damage [10,11]. In the present study, spectral changes of reflected light from denaturing tissues during the heating were investi- gated in vitro to monitor the thermal damage as a function of the temperature of the tissues. First, the basic theory of denaturation of biological tissues was illustrated. Then the experimental system to measure spectral distribution changes of light reflected from biological tissues for a varia- tion of temperature was explained. Results and analysis of Contract grant sponsor: American Society of Laser Medicine and Surgery Member Research Grant; Contract grant number: NIH F32 GM 066693-01. *Correspondence to: Hiroyuki Yoshimura, PhD, Department of Urban Environment Systems, Faculty of Engineering, Chiba University, 1-33, Yayoi-cho, Inage-ku, Chiba 263-8522, Japan. E-mail: yoshimura@faculty.chiba-u.jp Accepted 3 August 2005 Published online 27 September 2005 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/lsm.20240 ß 2005 Wiley-Liss, Inc.