ORIGINAL ARTICLE Year : 2011  Volume : 3  Issue : 2  Page : 266276 Artificial semirigid tissue sensitized with natural pigments: Effect of photon radiations Adnan Jaradat^{1}, MAli H AlAkhras^{1}, G Makhadmeh^{1}, K Aljarrah^{1}, A Alomari^{2}, Z Ababneh^{3}, Majed M Masadeh^{4}, HM AlKhateeb^{1}, BA Albiss^{1}, M Alshorman^{1}, ^{1} Department of Physics, BioMedical Physics Laboratory, Jordan University of Science and Technology (JUST), P.O. Box 3030, Irbid 22110, Jordan ^{2} Department of General and Pediatric Surgery, Faculty of Medicine, Jordan University of Science and Technology (JUST), P.O. Box 3030, Irbid 22110, Jordan, Jordan ^{3} Department Physics, Yarmouk University, Faculty of Pharmacy, Jordan ^{4} Department of Pharmaceutical Technology, Faculty of Pharmacy, Jordan Correspondence Address: Background: A new approach for evaluating the optical penetration depth and testing its validity with Monte Carlo simulations and KubelkaMunk theory is used for artificial semirigid tissue sensitized with natural pigments. Photodynamic therapy is a promising cancer treatment in which a photosensitizing drug concentrates in malignant cells and is activated by visible light at certain wavelength. Materials and Methods: Cheap artificial semirigid tissue incorporated with scattering and absorbing materials along with some other composites comparable to normal human tissue has been performed. The optical parameters as measured with different conditions and calculated with various techniques are investigated. Results: The probability of interaction of light with tissue is very high when exposed to light in presence of Cichorium pumilum and RBCs followed by photohemolysis or/and photodegradation. The optical penetration depth calculated by linear absorption coefficient ranges from 0.63 to 2.85 mm is found to be comparable to those calculated using KubelkaMunk theory or Monte Carlo simulation (range from 0.78 to 2.42 mm). The ratio of absorption to the scattering is independent of thickness and decreases with increasing irradiation time. Moreover, the optical parameters as well as their ratios are in very good agreement in the two approaches of calculation. The values of absorption and scattering coefficients are independent of thickness. Furthermore, the average photon ranges in the samples containing no scattering and absorbing materials are about three times greater than those samples containing scattering materials. Conclusion: Our results suggest that light propagation with optical properties presented in this work could be applicable in diagnostic and therapeutic of the human biological tissue for photodynamic therapy.
Materials and Methods In the onedimensional diffusion problem, we are interested in measuring the diffused reflectance R and transmittance T, in order to calculate the coefficients of absorption K and reduced scattering S '. The diffused transmittance and reflectance represent the amount of light that exits the rear and front surfaces of the sample, respectively. For the purpose of these measurements we used the integrating sphere, which provides a measured means of all transmittance or reflectance in the forward or backward hemispheres. The integrating sphere is a hollow sphere with an interior coated with highly diffused reflecting paint. The sample is placed in front of the sphere for transmittance measurements and behind the sphere for diffused reflectance measurements. The entering light is repeatedly reflected at the wall of the sphere until the light intensity at any point on the sphere is proportional to the intensity of the entering light. Light source The broadband light source used was a 200W highpressure Hg/Xe arc. The exposure light source was filtered by a Corning C.S. No. 052 filter (>360 nm) and 2 cm of water. The water filter was placed between the source of light and the sample to reduce heating. Energy fluence rate at the exposure site was about 60 J/cm 2 as measured by FiledMaxII Laser power/Energy Meter/Coherent/USA. The distance between the sample and the light source was 17 cm. The appropriate spot size of light source that covers the sample area was focused and collimated by a convex lens. Preparation of solutions PhosphateBuffered Saline (PBS) (Solution A): One pellet of PBS was dissolved with 200 ml of distilled water (SigmaAldrich p4417100AB, USA). A stirrer was used until the tablet completely dissolved. Cichorium pumilum (C.P.) A concentration of 10 mg/ml of the Cichorium pumilum (C.P.) has been prepared by dissolving 10 mg of the extracts with 1 ml of the PBS. Solution was exposed to ultrasonic action for 5 min with stirring continuously to completely dissolve the extract in the PBS solution. The concentration of C.P. in the samples is 2 mg/ml. Drawing ink solution (Solution B) Drawing Ink Solution was prepared by adding small amount of the ink to distilled water. The amount of the drawing ink was increased until the absorption factor reaches 0.18 at 500 nm wavelength. [34] The concentration of drawing ink in the samples is 0.02 mg/ml. Red blood cells Fresh blood samples from healthy volunteer donor were collected and washed out by PBS, pH 7.4. Mixture then left for 8 minutes spinning in a centrifugation 1500×g, at room temperature. This process was repeated at least three times to get colorless supernatants. Washed cells were diluted with PBS to get optical density equal to or less than 0.9 at 500 nm. [34] Preparation of the artificial tissue phantom A total volume of about 100 ml sample of similar optical properties of the esophageal wall in human was prepared: 2 grams of agar (that do not have an effect on the visible spectrum) had been dissolved in 40 ml of the PBS solution and 40 ml of distilled water. The solution was heated up to boiling point to make sure that the agar is dissolved totally. A stirrer with small magnet inside the sample was used to keep the sample homogenous. Solution was left to cool alone and free the bubbles out. As the solution cool down between (70 and 80°C), 3 g of silica powder is added of 45 micrometer in diameter to the solution and then the solution is placed in ultrasonic path for 5 min to discard the crusts of this material. At 45°C, 1 ml of solution B, 20 ml of bovine serum, and 0.2 ml of Intralipid is added, then the solution cooled down to 40°C. Finally, 1.5 ml of concentrated blood, photosensitive material (Cichorium Pumilum) on concentration of 2 mg/ml, and 1 ml of penicillin streptomycin were added. To obtain slices of the prepared artificial phantom, the glass slides of 0.85 mm empty inbetween gap were immersed inside the final solution. The separation gap was maintained by small magnet strips of 0.85 mm (sample thickness) between the upper and lower edges. The role of various composites in the phantom Six important materials were used in this experiment. Most of the concentrations and the method used in this work were reported by Bashkatov et al.[35] The concentration and the role of each composite are as follows: Ink and blood The Indian ink and blood are the optical absorbers. As different ink batches from the manufacturer may differ in their optical properties, Ink and blood were diluted to have absorbance value of 0.18 and 0.9, respectively, at λ =500 nm. Silica powder and the intralipid The silica powder and the Intralipid are responsible for light scattering. Silica Powder (FIZMERK an ISO 90012000 Certified Company, Fizmerk India Chemicals. India) prepared with concentration of 30 mg/ml, intralipid (SIGMAALDRICH, L141, 117k07251) with concentration of 0.2%. Bovine serum The role of the bovine serum is to have proteins in the phantom. The optical properties (absorption and scattering) of the serum do not play a significant role in the overall optical coefficients of the phantom. [35] (SIGMAALDRICH) Penicillin (SIGMAALDRICH p4333, 057k2411). The penicillin of 1% concentration in the sample was used to prevent contamination and insure the long time protection of such phantoms. [35] Agar (GAINLAND CHEMICAL COMPANY, 00310). Agar with a concentration of 20 mg/ml in the samples is a typical media used to contain all other materials to perform the artificial semirigid tissue. Agar was used as a reference and has been approved to have no significant effects on our reading due to its optical and chemical properties. Artificial tissue made of agar only was used as a background correction. [35] Phosphatebuffered saline (SIGMAALDRICH, p4417, 036k8200). Phosphatebuffered saline (PBS) with pH 7.4 is a substance that is used to maintain the red blood cells and to prevent the rupture of the Red blood cells (RBCs) membrane. Determination of the optical properties The objective of this research is to study the optical properties of the artificial tissue phantom. The main parameters measured in this work are absorption coefficient K and the scattering coefficient S. Moreover, the potential use of extracted photosensitizer C.P. is one of the main investigated goals. The variations of K and S with exposure time to light in the presence of C.P. were investigated. The variation of transmittance T with the thickness d has also been investigated. K/S, S, and K versus exposure time One sample with concentration (2.0 mg/ml) of C.P. and with thickness 0.85 mm was prepared. The spectra of R and T have been measured at different exposure times, and the values of K/S, S, and K were calculated by Monte Carlo simulation for each R and T. Variation of transmittance versus thickness In this part the transmittance has been measured with variation of the thickness at different wavelengths (400, 500, 600, 630 and 700 nm). The values of linear attenuation coefficient μatt and half value layer (HVL) at different wavelengths were measured. Five samples were prepared with fixed concentration of 2 mg/ml of C.P. The half value layer HVL and the attenuation coefficients μatt are plotted versus wavelength. Mathematical calculations Kubelkamunk theory KubelkaMunk theory is considered to be one of the most acceptable theories in optical tissue calculations. The most important and fundamental parameters in soft biological tissues are absorption coefficient K and scattering coefficient S. To calculate these values the reflectance (R) and transmittance (T) of the tissue must be measured. KubelkaMunk theory relates the scattering coefficient S and the absorption coefficient K directly with the measured reflectance (R) and transmittance T as follows: [INLINE:1] where d is the sample thickness and [INSIDE:1] The optical penetration depth (δ) can be calculated by using the values of S and K in the following wellknown equation. [35] [INLINE:2] where g is the average cosine of the scattering angle; the value of g has a maximum value of +1 for the entirely forward direction and has a minimum value of 1 for entirely backward scattering. The typical value of g for soft tissues varies from 0.7 to 0.9. Therefore, the reduced scattering coefficient is defined as S / = S [INLINE:3] BeerLambert Law: The differential decrease of light intensity in the medium due to absorption is proportional to the intensity I and elementary distance traveled by the light [INLINE:4] where: μ is the linear attenuation coefficient of absorber at incident wavelength. I is the intensity of light at depth d. I o is the intensity of light at depth d = 0. If we introduce the transmittance in terms of thickness we can rewrite BeerLambert Law as [INSIDE:2] New approach for calculating the optical penetration depth (δ): The optical penetration depth δ can be obtained from the experimental data based on BeerLambert's absorption law: [INLINE:5] This method of calculation depends on the assumption that the slope of the linear plots of ln(1/T) vs. d represents the effective linear attenuation coefficient. [35] Therefore, only measurements of transmittance (T) vs. thickness d is required. Two sets of samples were prepared to measure the absorption and scattering coefficients. The first set contains all the composites in the tissue phantom along with absorbers materials (ink + blood + C.P.) except the scattering materials (silica powder, intralipid). The slope ln(1/T) vs. d obtained from this set of samples is an effective linear absorption coefficient (μ a ). The second set contains all the composites in the tissue phantom along with scattering materials (silica powder, intralipid) except the absorbing materials (ink + blood). The slope of ln(1/T) vs. d obtained from this set of samples is an effective linear scattering coefficient (μ s ). [36] Therefore, effective attenuation coefficient (μ att ) inside the tissue could carry useful information about the tissue based on its composites. The effective attenuation coefficient is proportional to the density of the scattering particles. [37] Thus, the values of K and S can be replaced by μ a and μ s , respectively, and therefore equation (1) can be written as: [INLINE:6] Define the reduced scattering coefficient μ s' = μ s (1  g), therefore, equation (5) can be written as [INLINE:7] The validity of two methods of calculation was conformed. Results The interaction of light with matter in a single or multilayered system described by KubelkaMunk theory is the most acceptable model, which is based on measuring the diffuse reflectance and transmittance of the beam. The scattering and absorption coefficients, S and K, respectively, were calculated from the ratio of K/S. Another approach of calculating the optical penetration depth led to good agreement with those calculated by using KubelkaMunk theory. Plots of K/S, S, and K versus the exposure time single layer of tissue phantom The diffuse reflectance R and transmittance T were measured and the values K/S, S, and K were calculated by using Monte Carlo simulation. These values were plotted as a function of wavelength at different exposure times. [Figure 1] shows the variation of K/S with wavelength λ at different exposure times for time intervals of 0 min, 10 min, 25 min, 45 min, and 70 min. The flat portion of the curve starts at nearly 500 nm wavelength. The values of K/S at λ = 630 nm for unexposed sample (t = 0 min) is 1.2 and for exposed sample of exposure time of 70 min. K/S at λ = 630 nm is 0.7. As the exposure time increase the ratio K/S decrease.{Figure 1} The scattering coefficient S at 2.0 mg/ml of C.P. is shown in [Figure 2]. It shows the variation of S with wavelength λ at different exposure times of time intervals of 0 min., 10, 25, 45, and 70 min. Where S is maximum at λ = 500 nm. The maximum value for zero exposure time is 0.215 and for 70 min. exposure time is 0.189. {Figure 2} [Figure 3] shows the variation of K with wavelength λ by measuring the reflectance R and the transmittance T at different exposure times of time intervals of 0, 10, 25, 45, and 70 min. The curves of K change the direction of the gradients at 450 nm wavelength. The absorption coefficient decreases as the exposure time increases. {Figure 3} [Figure 4] represents the typical curves of absorption and scattering coefficients vs. wavelength at zero exposure time. {Figure 4} Plots of K/S, S, and K versus the exposure time for 2.0 mg/ml of C.P. with multilayer In this part, multilayer were used with samples incorporated with 2.0 mg/ml C.P. The values of transmittance and diffuse reflectance were measured and the value of K/S, S, K, were calculated. The variation of transmittance (T) with wavelength (λ): In this part we studied the variation of T vs. the wavelength at different tissue thicknesses. [Figure 5] illustrates the typical variation of transmittance beam T with different wavelength λ at different thickness for unexposed sample. (sample 4: Concentration = 2.0 mg/ml of C.P, 1 ml of drawing ink, 1.5 ml of red blood cells, 3 g of silica powder, 0.2 ml Intralipid). {Figure 5} Linear attenuation coefficient (μatt) and halfvalue layer (HVL) versus wavelength: The optical penetration depth of tissue when C.P., Scatter, Absorber are incorporated individually or together in the tissue phantoms were investigated. All photobiology are initiated by light interaction with tissue. The nature of interactions and photo effects depend on the biological and optical properties of the tissue as well as the power of the light. Light is strongly attenuated inside the tissue. In addition to T and R, two processes of interaction might take place inside tissue during attenuation; 1  Absorption which converts the energy from one form to another, such as heat, luminescence, and/or chemical energy. 2  Light scattering which changes the direction of the light rays without any loss of intensity. Half value layer (HVL) is the depth of tissue at which the beam intensity decays to half of its initial value. In [Figure 6], ln(T) has been plotted vs. the tissue thickness d in a semilog scale, the dot points represent the values of experimental data and the lines represent the fitting of these data to a straight line equation. At T = 0.5 the values of HVL are obtained at different wavelengths. We have found that the HVL in the tissue phantom is increasing with increasing the wavelength. Also from the same figure [Figure 6], we have calculated the effective linear attenuation coefficient (μatt). We have found that the value of (μatt) is decreasing with increasing the wavelength; data are shown in [Table 1]. {Figure 6}{Table 1} [Figure 7] shows the variation of wavelengths vs. the linear attenuation coefficient μatt and the half value layer HVL for the 2.0 mg/ml sample. {Figure 7} The reciprocal of 1/μ att is approximately equal to the penetration depth in which a collimated light beam is attenuated. The actual penetration of light inside tissue is deeper than 1/μ att due to forward scattering beam. [Table 1] and [Table 2] review the values of effective linear attenuation coefficients with different irradiation times for different composites. The optical penetration depth has been calculated on the bases of KubelkaMunk theory using Eq. 1. Another approach for calculating the optical penetration depth can be approximated by using Eq. 5 where the values of μαand μ s are used instead of K and S. The value of (μα) is the fraction of radiation absorbed for different sample contents per unit thickness of the absorber and (μ s ) is the linear scattering coefficient defined as the factor that expresses the attenuation caused by scatter materials. In this part we have calculated μα of the absorber composites (Blood and Ink) incorporated with the phantom tissue and μ s were calculated for the scatter composites (Silica powder and Intralipid) incorporated with phantom tissue, and also we have calculated the linear attenuation coefficient μ Agar of the agar material. The comparisons between the two approaches with other parameters are listed in [Table 2]. {Table 2} Figure shows a plot of ln(1/T) with tissue thickness (d) for the tissue phantoms at specific wavelength (λ = 630 nm) to show the linearity of ln(1/T) as a function of depth. [Table 3] illustrates the values of the ratio K/S for thick samples of d=4.25 mm. The calculation is based on the assumption that the sample is thick enough so that the transmission of the light as shown in [Figure 5] {Table 3} is negligible (0 to 7%). The values of K/S calculated from the measured values of R and T are in good agreement with the values calculated from the measured value of R only and T=0. Discussion Calculated S and K at different C.P. concentration with one layer Most biological tissues are very nonhomogeneous and the probability of scattering and absorption in spite of transmission and reflection are still considered to be very high. The distribution of light and light propagation inside biological tissue will remain ambiguous and subject of interest. The penetration of light inside any biological tissue can be expressed in terms of radiation passes through the tissue. Penetration is the inverse of attenuation. Generally speaking, increasing photon irradiation decreases the probability of interaction (attenuation) and hence increasing the probability of penetration. Therefore, all the photons that have traveled a few tenth of a millimeter into the tissue subjected to multiple scattering and will reach a molecule from all directions. These results are almost isotropic light distributions in the region distal to the surface, thus allowing the use of another approach instead of diffusion theory in describing the propagation of light inside the tissue. Scattering of light by particles inside most of the tissues occurred when the wavelength of light are comparable to or greater than the dimensions of the composites. When light photons are scattered by molecules in the medium, the energy of the photon is not changed significantly. The values of K/S related to the samples incorporated with 2.0 mg/ml of photosensitizer C.P. show a rapid decrease with increasing wavelength from 400 nm up to nearly 450 nm, followed by constant plateau shape from about 500 nm to 700 nm [Figure 1]. [Figure 2] represent the sample containing 2.0 mg/ml of C.P. that shows a slight reduction of scattering coefficient at the maximum peaks (range 0.22 to 0.19 mm 1 ) and the values of maximum peaks decreases with increasing irradiation time. The values of scattering coefficient calculated from the transmission found by (Wei Sun, et al., 1992) with phantoms incorporated with different concentration of Intralipid are (0.991.73 mm 1 ) which is comparable to those calculated by diffuse reflectance range (0.981.63 mm 1 ). The values of K related to the samples incorporated with 2.0 mg/ml of photosensitizer C.P. show rapid decrease by increasing the wavelength from 400 nm up to nearly 475 nm [Figure 3]. The curves followed by almost constant plateau shape with slight decrease from about 500 nm to 700 nm. Interestingly, at 630 nm it has been found that the ratio K/S decreases with increasing irradiation time and decreases with decreasing K and S. Calculation of S and K with multilayer The values of K/S are independent of thicknesses at different wavelengths while the values of K/S decrease with increasing irradiation time [Table 4]. The values of S are found to be independent of thickness and irradiation time [Table 5]. In contrast; with the case of onelayer samples the values are dependent on irradiation time, and that could be attributed to homogeneity of the one thin layer. The values of K are found to be independent of thickness but dependent on irradiation time [Table 6]. Similar approach by Kumar et al. reports that the emergence of the backscattered radiation from the deeper layers at various locations of the surface from the beam entry point is unaffected by the optical parameters of the tissues.{Table 4}{Table 5}{Table 6} The variation of transmittance T with wavelength for multilayer samples: The typical curves of transmittance T vs. wavelength λ and thickness d are shown in [Figure 2]. The slight fluctuations shown in the curves could not be defiantly clarified, but most probably could be due to the inhomogeneous tissue phantom or inhomogeneous light beam passes through the tissue phantom. [38] A beam with higher (m) would be more likely to interact and could have a smaller HVL and its intensity is reduced and this can be seen clearly in [Figure 8]. Previous works show similar plots of Half Value Layer (HVL) and attenuation coefficient μ where HVL increases with decreasing μ. [39] {Figure 8} The optical penetration depth in all biological tissues at 630 nm ranges from 0.1 to 3 mm. [40],[41] The optical penetration depth has been calculated on the bases of KubelkaMunk theory using Eq. 1. Another approach for calculating the optical penetration depth can be approximated by using Eq. 5 where the values of μαand μ s are used instead of K and S. The value of (μα) is the fraction of radiation absorbed for different sample contents per unit thickness of the absorber and (μ s ) is the linear scattering coefficient defined as the factor that expresses the attenuation caused by scatter materials. In this part we have calculated μα of the absorber composites (Blood and Ink) incorporated with the phantom tissue, and μ s is calculated for the scatter composites (Silica powder and Intralipid) incorporated with phantom tissue, and also we have calculated the linear attenuation coefficient μ Agar of the agar material. The comparisons between the two approaches; values of the optical penetration depth using Eq. 1 and values of the optical penetration depth using Eq. 5 are in very good agreement [Table 2]. Furthermore, the values of 1/μ att represent the average range defined as the distance traveled by the photons inside the tissue phantom before interaction takes place. Photon ranges are increasing with increasing wavelength [Table 2]. The average range of a group of photons is inversely related to the effective linear attenuation coefficient. The average range of photon ranges for samples containing ink, blood, agar, penicillin, bovine serum and C.P. is from 1.613 to 6.135 mm, while for samples contain silica powder, Intralipid, agar, penicillin, bovine serum and C.P. is 1.294 to 2.288 mm, for samples contain ink, blood, silica powder, Intralipid, agar, penicillin, and bovine serum (without C.P) is from 1.587 to 1.949 mm. Finally, for samples containing agar, penicillin, and bovine serum only the photon range is 3.906 to 7.143 mm. As expected the average photon range in samples containing no scattering and no absorbing materials are about three times greater than those containing scattering materials. Similarly with samples containing absorbing materials (without scattering materials) are comparable to those containing agar, penicillin, and bovine serum only. Furthermore, the validity of our results can be seen from the good agreement between the values of K/S (calculated from the measured values of R and T) with the values calculated from the measured value of R only (assuming T is zero) [Table 3]. Conclusions The optical parameters measured at various conditions and calculated by various technique represent the behavior of light propagation in tissue phantom. The probability of interaction of light with tissue is very high when exposed to light in present of C.P. and RBCs followed by photohemolysis or/and photodegradation. Therefore, the values of absorption coefficient decrease from 0.65 to 0.13 mm 1 . The conclusions of this work can be summarized as follows: The values of K/S show rapid decrease with increasing wavelength from 400 nm up to nearly 450 nm, followed by constant plateau shape from about 500 nm to 700 nm. The values of K/S are independent of thickness and the values of K/S decrease with increasing irradiation time. The values of K are independent of thickness and depend on irradiation time. The values of S are independent of thickness and irradiation time. The average photon range in the samples contains no scattering and absorbing materials are about three times greater than those samples containing scattering materials. HVL in the tissue phantom is increasing with increasing wavelengths. The value of (μ att ) is decreasing with increasing the wavelength. Light propagation with the optical properties presented in this work could be applicable in diagnostic and therapeutic of human biological tissue for photodynamic therapy. Very good agreement between the two approaches of calculating the optical penetration depth. Acknowledgments This work was funded by the Scientific Research Faculty, grant # 802008 and partially by higher council for science and technology/Jordan., grant # 62/2000. References


