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Depolarization of light in biological tissues
Optics and Lasers in Engineering 50 (2012) 850–854
Contents lists available at SciVerse ScienceDirect
Optics and Lasers in Engineering journal homepage: www.elsevier.com/locate/optlaseng
Depolarization of light in biological tissues Dror Fixler a,n, Rinat Ankri a, Hamootal Duadi a, Rachel Lubart b,1, Zeev Zalevsky a a b
Faculty of Engineering and the Institute of Nanotechnology and Advanced Materials, Bar Ilan University, Ramat Gan 52900, Israel Department of Chemistry, Bar Ilan University, Ramat Gan 52900, Israel
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 November 2011 Received in revised form 12 December 2011 Accepted 13 January 2012 Available online 2 February 2012
Recently in phototherapy the use of diodes and broadband light devices instead of lasers was suggested for economical and practical reasons. It has been argued that lasers are not superior to LEDs since they lose their coherence and polarization once they penetrate into biological tissues. However, the polarization point has never been experimentally proven. In this work, to the best of our knowledge, we have for the first time experimentally validated the conditions that affect the polarization state of light when laser illumination propagates through a biological tissue with and without flow. In our experiments we measured the polarization of light passing through phantoms as well as through uncooked turkey meat. The measurements were performed for varied integration time, thickness and flow rates. It was experimentally validated that the tissue thickness hardly influences the polarization in comparison to flow for a reduced scattering coefficient of 0.8 mm 1 while there is no flow. Furthermore, when the flow is perpendicular to the polarization plane its velocity highly affects the polarization. However, when the flow is parallel to the polarization plane there is almost no change in the propagating light’s polarization state. Thus, one outcome of this work is that since the biological tissue is not static and contains many blood vessels and capillaries, the polarization of the laser may be lost when light penetrates the tissue. & 2012 Elsevier Ltd. All rights reserved.
Keywords: Flow Phantoms Polarization Tissues Multi-Scattering
1. Introduction The use of non-polarized light instead of polarized lasers in biomedical applications such as phototherapy was recently suggested for economical and practical reasons. In many, although not all, cases the output of a laser is polarized. This normally means a linear polarization state, where the electric field oscillates in a certain direction perpendicular to the propagation direction of the laser beam [1]. It has been argued that lasers are not preferred, as the latter lose their polarization direction once they penetrate into static biological tissues, all the more once they penetrate into flowing medium. Light–tissue interaction is common in clinical treatments and in medical research as different light sources in the visible and the near infrared (NIR) spectral ranges are widely used. Especially, in the therapeutic field, such as photodynamic therapy that uses light to damage tumor cells while light activates a photochemical that induces the formation of reactive oxygen species that induces damage, and in Low Level Laser Therapy (LLLT) that uses visible– NIR light to bio-stimulate cells [2–5]. LLLT has been used during the last years for the treatment of various pathological conditions,
n
Corresponding author. Tel.: þ972 3 5317598; fax: þ972 3 7384051. E-mail address: dror.fi[email protected] (D. Fixler). 1 Tel.: þ972 3 5317797; fax: þ972 3 7384053.
0143-8166/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.optlaseng.2012.01.011
such as musculoskeletal injures, arthritis, chronic wounds, etc. Light phototherapy uses monochromatic light (lasers), quasimonochromatic radiation (LEDs) and broadband light devices. The laser differs from other light sources in its polarization characteristics. The growing acceptance of LEDs and broadband light devices (which are non-polarized light sources) in phototherapy puts into debate the value of polarization for enhancing the benefit from the light associated biomedical treatment. A widely discussed issue in the laser phototherapy clinical community is the question whether the coherence of laser radiation has additional benefits, compared to monochromatic light originated from a conventional light source or LED of the same wavelength and intensity that is not polarized by additional filters. Our previous research deals with the preservation of the coherence state while propagating through a tissue with flow [6]. There we have found that the laser coherence is partially lost in proportion to the fluid’s flow rate through the tissue. In this paper we intend to investigate the preservation of the polarization state for light propagating through a tissue with and without flow. Specially designed experiments in the cellular level have presented that He–Ne laser radiation (i.e., coherent and polarized light at 632.8 nm) and properly filtered light (633 nm) from a conventional light source, with the same intensity and irradiation time, have the same biological effect [7]. The same conclusion was drawn from an experiment in which radiation from a He–Ne laser
D. Fixler et al. / Optics and Lasers in Engineering 50 (2012) 850–854
was used with or without an optical fiber for full depolarization of the beam [8]. To further evaluate the importance of polarization for the stimulation of cellular metabolism, Karu et al. [9] studied a possible dependence of the level of light polarization (99.4%, 60.9% and 34.2% linearly polarized light at 637 nm) on the model of cell attachment to a glass matrix. They concluded that elementary processes such as stimulation of cell attachment do not depend on the light polarization level. However, this conclusion is true for a cellular suspension and might differ from real bio-tissues. Two other in-vivo works (in animal) claimed different effects of polarized and non-polarized light. Chumak et al. [10] found that the irradiation of rat hind limb with polychromatic polarized light at 400–2000 nm produced a long-term inhibition of baseline afferent traffic in the saphenous nerve, while non-polarized light produced biphasic changes in baseline activity. Ribeiro et al. [11] reported a very interesting effect concerning light polarization. They investigated the healing of burns in rats by irradiating the lesions with a linearly polarized He–Ne laser beam parallel to the rat’s spinal column and using the same laser and dose, but aligning the light polarization perpendicularly to the relative orientation of the spinal column. They found that the skin wound repair depended on polarization orientation with respect to a reference axis of the animal’s spinal column. At the clinical level, there are a lot of studies [12–18] presenting successful polarized light therapy at 400–2000 nm, 40 mW/cm2, 2.4 J/cm2, for wound healing, pain relief and more. Unfortunately, almost all of these studies did not compare their results with the same device having non-polarized radiation. Only Zhevago et al. [19] performed a randomized, placebo-controlled double-blind trial study with the same polarized and non-polarized light source. They measured changes in the humeral immunity of a large group of volunteers after exposure of a small body area to polychromatic visible and infrared polarized (VIP) and non-polarized (VInP) light (400–3400 nm, 95% polarization, 40 mW/cm2, 12 J/cm2 and 400–3400 nm, no polarization, 38 mW/cm2, 11.2 J/cm2). They demonstrated high similarity between the effects induced by a single exposure of both light sources. Another type of static measurement that found a difference between polarization directions at in-vivo measurement was reported 20 years ago [20]. Anderson separated light reflected from skin into two components: regular reflectance or ‘‘glare’’ arising from the surface and light backscattered from within the tissue. He found that when the planes of polarization are parallel, images with enhanced surface details are obtained. However when the planes are orthogonal, wrinkles and surface details disappear and an enhanced view of vasculature and pigmented lesions is obtained. Although it is known that polarized light is rapidly scrambled in highly scattering media such as biological tissues (probably in the first few hundred mm) [21,22], it seems very unlikely that polarization could play a role in any significant applications for the upper skin layers. The ability of polarized light to aid tissue imaging has a long history that was summarized by Jacques et al. [23]. Sankaran et al. [24,25] studied the influence of densely packed microsphere solution on the transmitted polarized beam, demonstrating that the behavior of polarized light in tissues is similar to the behavior in densely packed microsphere solutions. A few years later they showed that circularly polarized light is always less depolarized than linearly polarized states unless the relative degree of polarization values of both polarizations highly depends on the scattering regime, which is typical for the Mie regime and multiple scattering [26]. However, further experiments with bio-tissues are needed in order to acquire higher understanding of the possible role of the polarization of light in phototherapy. In the present study we experimentally tested the loss of polarization in tissues with and without flow.
851
In Section 2 we describe the materials and methods of the measurement system. In Section 3 we perform the experimental measurements and present the obtained results. Section 4 concludes the paper.
2. Materials and methods In this research we built an experimental set-up for polarization measurement of the light being transmitted through phantoms containing intralipid and ink components [27] as well as uncooked turkey meat. Solid phantoms with different structures and optical properties (absorption coefficient range of 0.0064– 0.03 mm 1, scattering coefficient range of 2–3 mm 1 and anisotropy factor of g¼0.72) were prepared in order to simulate different human tissues. The turkey meat’s reflected light intensity profile was compared and found to be similar to those of the phantoms [6]. The phantoms were made using ink 0.1%, as an absorbing component, intralipid 20% (IL, Lipofundin MCT/LCT 20%, B. Braun Melsungen AG, Germany), as a scattering component and agar powder, in order to convert the solution into gel. The phantoms were prepared in cell culture plates (90 mm) and were cooled in vacuum conditions (to avoid bubbles). The phantoms’ optical properties are presented in Table 1. The reduced scattering coefficient ms0 (ms0 ¼ ms(1 g)) that we used was relatively small (0.8 mm 1) in order to preserve the polarization of the propagating light in the static measurements. Typically, one chooses a phantom by matching its scattering and absorption properties, to those of the tissue. However, this does not necessarily imply that the polarization properties of the phantom match those of the tissue [28], which is the goal in our experiments. Note that a recent work showed that such intralipid based phantoms, as used in our study, highly imitate depolarization tissue properties [29]. The experimental system includes (Fig. 1a) a green doubled Nd:YAG laser at a wavelength of 532 nm as an excitation source that was used to illuminate the sample after passing through a polarizer. The light that passes through the sample is captured using a Pixelink PL-A741-E camera (PixeLINK, Ottawa, ON) with a pixel size of 7 mm 7 mm after passing through a second polarizer. The distance between the excitation polarizer and the flowing medium was 6 cm and the detector was focused on the emission polarizer (real distance of 2.5 cm). The camera lens’ numerical aperture (NA) was 0.2. The optical setup diagram (Fig. 1b) illustrates that since the illumination is scattered by the sample, there is a need for a camera lens in order to collect the light to the CCD detector. The flows inside the phantoms were generated using needles with two different diameters of G21 and G22 (G-Gauge; resulting in 0.8128 mm and 0.7112 mm, respectively). The actual diameter of the tunnel in the phantom and the tissue was verified using an optical microscope (Olympus BX51) once the needles were withdrawn. The tunnels inside the samples (phantoms as well as the turkey meat) were created by needles and water was injected to the tunnels by a 10 ml syringe. In order to get different flow Table 1 Optical properties of the irradiated phantoms. The concentration of IL refers to the fraction of solids in the solution, while the concentration of ink pertains to the fraction of the original product. Phantom #
Optical properties [mm 1]
Ink concentration [%]
IL concentration [%]
1 2 3 4
m ¼0.0064, ms0 ¼0.8 m ¼0.0073, ms0 ¼0.8 m ¼0.0096, ms0 ¼0.8 m ¼0.0192, ms0 ¼0.8
2 10 4 2.2 10 4 3 10 4 6 10 4
0.8 0.8 0.8 0.8
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D. Fixler et al. / Optics and Lasers in Engineering 50 (2012) 850–854
particulates, the light’s polarization status changes. For this reason, first we measured the polarization of light that passed through the scattering and absorption medium of the static phantoms. We tested different types and widths of phantoms. The phantoms were prepared as described in Section 2 and four different phantoms that are presented in Table 1 were measured. The samples were prepared in order to mimic the optical properties of real biological tissues. Each sample was measured for varied widths of 1, 2, 4 and 5 mm. In Fig. 2 one can see representative results for the 4 different phantoms (from Table 1). The polarizers were positioned perpendicularly to one another, so that when no sample was inserted between them no light was collected by the camera and the polarization state (the normalized intensity along the direction of the output polarizer) remained 1 (marked as sample #0 in Fig. 2). When inserting the samples (1, 2, 4 and 5 mm wide phantoms with the characteristics described in Table 1), one could see a small reduction in the polarization state, but in all cases the polarization remained higher than 0.92. These results are in high agreements with previous reports [23,24]. 3.2. Polarization of real tissues
Fig. 1. Image (a) and optical diagram (b) of the experimental setup. A laser illuminates a sample after passing through the left polarizer and the transmitted light is imaged by a camera after passing through the right polarizer.
velocities, we used 1, 1.5 and 2 ml of tap water, purified water, PBS (pH¼7.4) and Dulbecco’s modified Eagle’s medium (including Calcium, Magnesium, Potassium and Sodium), which were injected to the tunnels by an engine bundled to different syringes, where the content of the syringe was injected to the sample during the same period of time (10 s). The scattering properties of the tap water, purified water and PBS were zero; the optical properties of Dulbecco’s modified Eagle’s medium were not measured directly but before starting the flow, the first control measurement was done including the sample and the medium without flow. We planned the above flow velocities (6–12 ml/s), volumes and tunnel diameters in order to achieve typical human blood flow velocities. The blood flow in the entire human circulation is about 5000 ml/min in an average sized adult at rest, but may be 5–6 times greater during heavy exercise [30]. The blood vessel diameters change from a few mm down to microns, so our rates and diameters are similar to the neck vein blood flow velocity [31]. By the term of polarization we refer in this research to the following ratio of measured intensities: P¼
Solid phantoms composed of ink and interlipid suspended in water may have bulk optical properties similar to those of a tissue (absorption and scattering coefficients, anisotropy of scattering, etc), but one may claim that they will not likely recapitulate tissue properties. The reason might be that the nature of the refractive index variation in the phantom is caused by discrete particulates, while tissue is a random continuum. In addition, it does not necessarily imply that the polarization properties of the phantom match those of the tissue [28]. In order to test this issue, measurements of real tissues (pieces of uncooked turkey meat) were performed and compared to phantom samples with similar optical properties [32]. The measurements were done for different widths of tissues and phantoms (1–4 mm). As can be seen from Fig. 3, the polarization properties in these tissues are identical to those measured in phantoms. Since scattering in static tissue structures rapidly depolarizes an incident beam, we have chosen to use samples whose polarization state remained high ( 40.9) without flow and only the flow depolarization takes effect. 3.3. Flow inside the phantoms Measurements of flow in phantoms were done as follows: phantoms with the same characteristics as were used for the
I0,0 I0,90 I0,0 I0,90
where Ia,b is the intensity measured when the first polarizer was positioned at an angle a and the second at an angle b. The captured videos of the transmitted light were stored as avi files and analyzed using the MATLAB (The MathWorks, Natick, MA) software. In particular, avi movies were translated to individual frames where each one of them contains all the image information, such as its time and exposure duration.
3. Experimental results 3.1. Static phantom As polarized light propagates through light-scattering media, such as biological tissues, microsphere solutions or an atmosphere with
Fig. 2. Polarization measured by the experimental setup for different phantoms without flow. Four different phantoms with 4 mm width are presented and their optical properties are described in Table 1. The polarization of all samples is larger than 0.92.
D. Fixler et al. / Optics and Lasers in Engineering 50 (2012) 850–854
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Fig. 3. Polarization for phantoms (diamonds) and for a piece of turkey meat (square) with different widths. Note that both samples have a contrast close to one, similarly to what was obtained in Fig. 2.
static phantom measurements and with 5 mm width were used for the flow measurements. Two different samples were prepared: one sample with a single tunnel of 0.8128 mm in diameter and another sample with a single tunnel of 0.7112 mm in diameter. The tunnels were generated in the middle of the samples. Four types of liquids (tap water, purified water, PBS and Dulbecco’s modified Eagle’s medium) at flow rates of 0.1 ml/s, 0.15 ml/s and 0.2 ml/s were injected into the tunnels. The flows were in different directions relative to the excitation polarizer. We measured flow in the same direction as that of the polarizer excitation, in a perpendicular direction to the polarizer and at 451. The results for the polarization state obtained for the purified water are presented in Fig. 4. When the polarizer and the flow were in the same direction there was almost no decrease in the polarization (still higher than 0.9) detected for all flow rates (black lines). When the flow was in a perpendicular direction to the light, the polarization state decreased up to 0.4 (green lines). For different flow rates we got different integration durations that generated a reduction of the polarization; for a flow rate of 0.1 ml/s in the perpendicular direction the polarization was reduced after 2 s to 0.6 (full thin green line), while for 0.2 ml/s flow velocity in the perpendicular direction the polarization reduced after 2 s to its final value of 0.4 (full thick green line). When the flow was at 451, we got the polarization values between the last two cases (red lines). For the other three types of liquids (tap water, PBS and Dulbecco’s modified Eagle’s medium) the results were the same (data not shown). These results, dealing with the different orientation of the polarizer relative to the flow direction, match a mathematical relation of cos2 y between the intensity in the perpendicular direction and the relative angle, meaning that the flow will not influence the polarization when it is in the same direction as the incident light’s polarization. However, when there is an angle of 901 between the polarization and the flow direction, the depolarization effect will be significantly stronger and at 451 the polarization is reduced by half. While at 01 we measure P¼ 0.9, at 901 P¼0.4; hence DP¼P(y ¼01)—P(y ¼901)¼0.5 and P(y ¼ 451)¼0.65 is exactly DP cos2 451þ P(y ¼901).
4. Conclusions and discussions The presented paper tries to understand the possible role of polarization of light in phototherapy. The biological tissue is not static and contains blood vessels and capillaries, where liquid is
Fig. 4. Polarization for phantom with different flow conditions. Three different flow rates were measured for three different orientations of the flow-polarizer. The full thin lines present a flow velocity of 0.1 ml/s; the dashed lines present a velocity of 0.15 ml/s; the full thick lines present a velocity of 0.2 ml/s; the black lines present flow in the same direction of the polarizer; the red lines present flow at 451 to the polarizer; the green lines present flow perpendicular to the direction of the polarizer. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
flowing in many directions. Therefore, we measured the polarization of light propagating through a biological tissue versus the volumetric flow rate and the integration time as well as the flow direction. We found that the polarization of the laser is almost uninfluenced by light passing through a static tissue with a low reduced scattering coefficient of ms0 ¼0.8 mm 1 or when the flow direction is parallel to the polarization direction of the laser. However, it is partially lost when there is a flow of fluid through the tissue in a direction perpendicular to the laser’s polarization direction. No evidence for optical activity was found. It should be noted that in the current study the tissues that we used was fresh flash tissues, which did not mimic the actual in vivo situation where blood is flowing within the living tissue. The volumetric flow rate is directly correlated to the polarization loss. Higher flow rate produces lower polarization values. This may happen due to the flow itself as well as due to the movements of the scattering elements inside the tissues, caused by the flow. The multi-scattering phenomena reduced the polarization value. The fact that the blood vessels in the body do not range in one direction means that the laser beam necessarily hits the fluid flow through the tissue perpendicular to its polarization direction. It can thus be concluded that from the polarization aspect – based on the fact that linear polarization of laser radiation after passing fluid media may be lost, there are no additional benefits in using it as an excitation source without maintaining the excitation direction; for example it may simplify pulse oximetry instrumentation. Furthermore, the change in polarization as a flow rate indicator could be used as a diagnostic tool for congestive heart failure. It might also follow wound healing rates in diabetic patients.
References [1] Lin JF, Lo YL. Measurement of optical rotation and phase retardance of optical samples with depolarization effects using linearly and circularly polarized probe lights. Opt Laser Eng 2009;47:948–55.
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[2] Ben Dov N, Shefer G, Irintchev A, Wernig A, Oron U, Halevy O. Low-energy laser irradiation affects satellite cell proliferation and differentiation in vitro. Biochim Biophys Acta 1999;1448:372–80. [3] Yu W, Naim JO, Lanzafame R. The effect of laser irradiation on the release of bFGF from 3T3 fibroblasts. Photochem Photobiol 1994;59:167–70. [4] Krauss JM, Puliafito CA. Lasers in ophthalmology. Lasers Surg Med 1995;17:102–59. [5] Reddy GK. Photobiological basis and clinical role of low intensity lasers in biology and medicine. J Clin Laser Med Surg 2004;22:141–50. [6] Fixler D, Duadi H, Ankri R, Zalevsky Z. Determination of coherence length in biological tissues. Lasers Surg Med 2011;43:339–43. [7] Karu TI, Kalendo GS, Letokhov VS, Lobko VV. Biostimulation of HeLa cells by low intensity visible light. Nuovo Cimento 1982;9D:828–40. [8] Bertoloni G, Sacchetto R, Baro E, Ceccherelli F, Jori G. Biochemical and morphological changes in Escherichia coli irradiated by coherent and noncoherent 632.8 nm light. J Photochem Photobiol B 1993;18:191–6. [9] Karu TI, Pyatibrat LV, Moskvin SV, Andreev S, Letokhov VS. Elementary processes in cells after light absorption do not depend on the degree of polarization: implications for the mechanisms of laser phototherapy. Photomed Laser Surg 2008;26:77–82. [10] Chumak AG, Chickan DN, Ulaschik VS. Effect of polychromatic polarized light on afferent traffic in skin branches of somatic nerves. Bull Exp Biol Med 2000;130:624–6. [11] Ribeiro MS, Da Silva DF, De Aranjo CE, De Oliveria SF, Pelegrini CMR, Zorn TMT, Zezell DM. Effects of low-intensity polarized visible laser radiation on skin burns: a light microscopy study. J Clin Laser Med Surg 2004;22:59–66. [12] Stasinopoulos D. The use of polarized polychromatic non-coherent light as therapy for acute tennis elbow/lateral epicondylalgia: a pilot study. Photomed Laser Surg 2005;23:66–9. [13] Monstrey S, Hoeksema H, Saelens H, Depuydt K, Hamdi M, Van Landuyt K, Blondeel P. A conservative approach for deep dermal burn wounds using polarized-light therapy. Br J Plast Surg 2002;55:420–6. [14] Medenica L, Lens M. The use of polarised polychromatic non-coherent light alone as a therapy for venous leg ulceration. J Wound Care 2003;12:37–40. [15] Monstrey S, Hoeksema H, Depuydt K, Van Maele G, Van Landuyt K, Blondeel P. The effect of polarized light on wound healing. Eur J Plast Surg 2002;24: 377–82. [16] Colic MM, Vidojkovic N, Jovanovic M, Lazovic G. The use of polarized light in aesthetic surgery. Aesthetic Plast Surg 2004;28:324–7.
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Contents lists available at SciVerse ScienceDirect
Optics and Lasers in Engineering journal homepage: www.elsevier.com/locate/optlaseng
Depolarization of light in biological tissues Dror Fixler a,n, Rinat Ankri a, Hamootal Duadi a, Rachel Lubart b,1, Zeev Zalevsky a a b
Faculty of Engineering and the Institute of Nanotechnology and Advanced Materials, Bar Ilan University, Ramat Gan 52900, Israel Department of Chemistry, Bar Ilan University, Ramat Gan 52900, Israel
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 November 2011 Received in revised form 12 December 2011 Accepted 13 January 2012 Available online 2 February 2012
Recently in phototherapy the use of diodes and broadband light devices instead of lasers was suggested for economical and practical reasons. It has been argued that lasers are not superior to LEDs since they lose their coherence and polarization once they penetrate into biological tissues. However, the polarization point has never been experimentally proven. In this work, to the best of our knowledge, we have for the first time experimentally validated the conditions that affect the polarization state of light when laser illumination propagates through a biological tissue with and without flow. In our experiments we measured the polarization of light passing through phantoms as well as through uncooked turkey meat. The measurements were performed for varied integration time, thickness and flow rates. It was experimentally validated that the tissue thickness hardly influences the polarization in comparison to flow for a reduced scattering coefficient of 0.8 mm 1 while there is no flow. Furthermore, when the flow is perpendicular to the polarization plane its velocity highly affects the polarization. However, when the flow is parallel to the polarization plane there is almost no change in the propagating light’s polarization state. Thus, one outcome of this work is that since the biological tissue is not static and contains many blood vessels and capillaries, the polarization of the laser may be lost when light penetrates the tissue. & 2012 Elsevier Ltd. All rights reserved.
Keywords: Flow Phantoms Polarization Tissues Multi-Scattering
1. Introduction The use of non-polarized light instead of polarized lasers in biomedical applications such as phototherapy was recently suggested for economical and practical reasons. In many, although not all, cases the output of a laser is polarized. This normally means a linear polarization state, where the electric field oscillates in a certain direction perpendicular to the propagation direction of the laser beam [1]. It has been argued that lasers are not preferred, as the latter lose their polarization direction once they penetrate into static biological tissues, all the more once they penetrate into flowing medium. Light–tissue interaction is common in clinical treatments and in medical research as different light sources in the visible and the near infrared (NIR) spectral ranges are widely used. Especially, in the therapeutic field, such as photodynamic therapy that uses light to damage tumor cells while light activates a photochemical that induces the formation of reactive oxygen species that induces damage, and in Low Level Laser Therapy (LLLT) that uses visible– NIR light to bio-stimulate cells [2–5]. LLLT has been used during the last years for the treatment of various pathological conditions,
n
Corresponding author. Tel.: þ972 3 5317598; fax: þ972 3 7384051. E-mail address: dror.fi[email protected] (D. Fixler). 1 Tel.: þ972 3 5317797; fax: þ972 3 7384053.
0143-8166/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.optlaseng.2012.01.011
such as musculoskeletal injures, arthritis, chronic wounds, etc. Light phototherapy uses monochromatic light (lasers), quasimonochromatic radiation (LEDs) and broadband light devices. The laser differs from other light sources in its polarization characteristics. The growing acceptance of LEDs and broadband light devices (which are non-polarized light sources) in phototherapy puts into debate the value of polarization for enhancing the benefit from the light associated biomedical treatment. A widely discussed issue in the laser phototherapy clinical community is the question whether the coherence of laser radiation has additional benefits, compared to monochromatic light originated from a conventional light source or LED of the same wavelength and intensity that is not polarized by additional filters. Our previous research deals with the preservation of the coherence state while propagating through a tissue with flow [6]. There we have found that the laser coherence is partially lost in proportion to the fluid’s flow rate through the tissue. In this paper we intend to investigate the preservation of the polarization state for light propagating through a tissue with and without flow. Specially designed experiments in the cellular level have presented that He–Ne laser radiation (i.e., coherent and polarized light at 632.8 nm) and properly filtered light (633 nm) from a conventional light source, with the same intensity and irradiation time, have the same biological effect [7]. The same conclusion was drawn from an experiment in which radiation from a He–Ne laser
D. Fixler et al. / Optics and Lasers in Engineering 50 (2012) 850–854
was used with or without an optical fiber for full depolarization of the beam [8]. To further evaluate the importance of polarization for the stimulation of cellular metabolism, Karu et al. [9] studied a possible dependence of the level of light polarization (99.4%, 60.9% and 34.2% linearly polarized light at 637 nm) on the model of cell attachment to a glass matrix. They concluded that elementary processes such as stimulation of cell attachment do not depend on the light polarization level. However, this conclusion is true for a cellular suspension and might differ from real bio-tissues. Two other in-vivo works (in animal) claimed different effects of polarized and non-polarized light. Chumak et al. [10] found that the irradiation of rat hind limb with polychromatic polarized light at 400–2000 nm produced a long-term inhibition of baseline afferent traffic in the saphenous nerve, while non-polarized light produced biphasic changes in baseline activity. Ribeiro et al. [11] reported a very interesting effect concerning light polarization. They investigated the healing of burns in rats by irradiating the lesions with a linearly polarized He–Ne laser beam parallel to the rat’s spinal column and using the same laser and dose, but aligning the light polarization perpendicularly to the relative orientation of the spinal column. They found that the skin wound repair depended on polarization orientation with respect to a reference axis of the animal’s spinal column. At the clinical level, there are a lot of studies [12–18] presenting successful polarized light therapy at 400–2000 nm, 40 mW/cm2, 2.4 J/cm2, for wound healing, pain relief and more. Unfortunately, almost all of these studies did not compare their results with the same device having non-polarized radiation. Only Zhevago et al. [19] performed a randomized, placebo-controlled double-blind trial study with the same polarized and non-polarized light source. They measured changes in the humeral immunity of a large group of volunteers after exposure of a small body area to polychromatic visible and infrared polarized (VIP) and non-polarized (VInP) light (400–3400 nm, 95% polarization, 40 mW/cm2, 12 J/cm2 and 400–3400 nm, no polarization, 38 mW/cm2, 11.2 J/cm2). They demonstrated high similarity between the effects induced by a single exposure of both light sources. Another type of static measurement that found a difference between polarization directions at in-vivo measurement was reported 20 years ago [20]. Anderson separated light reflected from skin into two components: regular reflectance or ‘‘glare’’ arising from the surface and light backscattered from within the tissue. He found that when the planes of polarization are parallel, images with enhanced surface details are obtained. However when the planes are orthogonal, wrinkles and surface details disappear and an enhanced view of vasculature and pigmented lesions is obtained. Although it is known that polarized light is rapidly scrambled in highly scattering media such as biological tissues (probably in the first few hundred mm) [21,22], it seems very unlikely that polarization could play a role in any significant applications for the upper skin layers. The ability of polarized light to aid tissue imaging has a long history that was summarized by Jacques et al. [23]. Sankaran et al. [24,25] studied the influence of densely packed microsphere solution on the transmitted polarized beam, demonstrating that the behavior of polarized light in tissues is similar to the behavior in densely packed microsphere solutions. A few years later they showed that circularly polarized light is always less depolarized than linearly polarized states unless the relative degree of polarization values of both polarizations highly depends on the scattering regime, which is typical for the Mie regime and multiple scattering [26]. However, further experiments with bio-tissues are needed in order to acquire higher understanding of the possible role of the polarization of light in phototherapy. In the present study we experimentally tested the loss of polarization in tissues with and without flow.
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In Section 2 we describe the materials and methods of the measurement system. In Section 3 we perform the experimental measurements and present the obtained results. Section 4 concludes the paper.
2. Materials and methods In this research we built an experimental set-up for polarization measurement of the light being transmitted through phantoms containing intralipid and ink components [27] as well as uncooked turkey meat. Solid phantoms with different structures and optical properties (absorption coefficient range of 0.0064– 0.03 mm 1, scattering coefficient range of 2–3 mm 1 and anisotropy factor of g¼0.72) were prepared in order to simulate different human tissues. The turkey meat’s reflected light intensity profile was compared and found to be similar to those of the phantoms [6]. The phantoms were made using ink 0.1%, as an absorbing component, intralipid 20% (IL, Lipofundin MCT/LCT 20%, B. Braun Melsungen AG, Germany), as a scattering component and agar powder, in order to convert the solution into gel. The phantoms were prepared in cell culture plates (90 mm) and were cooled in vacuum conditions (to avoid bubbles). The phantoms’ optical properties are presented in Table 1. The reduced scattering coefficient ms0 (ms0 ¼ ms(1 g)) that we used was relatively small (0.8 mm 1) in order to preserve the polarization of the propagating light in the static measurements. Typically, one chooses a phantom by matching its scattering and absorption properties, to those of the tissue. However, this does not necessarily imply that the polarization properties of the phantom match those of the tissue [28], which is the goal in our experiments. Note that a recent work showed that such intralipid based phantoms, as used in our study, highly imitate depolarization tissue properties [29]. The experimental system includes (Fig. 1a) a green doubled Nd:YAG laser at a wavelength of 532 nm as an excitation source that was used to illuminate the sample after passing through a polarizer. The light that passes through the sample is captured using a Pixelink PL-A741-E camera (PixeLINK, Ottawa, ON) with a pixel size of 7 mm 7 mm after passing through a second polarizer. The distance between the excitation polarizer and the flowing medium was 6 cm and the detector was focused on the emission polarizer (real distance of 2.5 cm). The camera lens’ numerical aperture (NA) was 0.2. The optical setup diagram (Fig. 1b) illustrates that since the illumination is scattered by the sample, there is a need for a camera lens in order to collect the light to the CCD detector. The flows inside the phantoms were generated using needles with two different diameters of G21 and G22 (G-Gauge; resulting in 0.8128 mm and 0.7112 mm, respectively). The actual diameter of the tunnel in the phantom and the tissue was verified using an optical microscope (Olympus BX51) once the needles were withdrawn. The tunnels inside the samples (phantoms as well as the turkey meat) were created by needles and water was injected to the tunnels by a 10 ml syringe. In order to get different flow Table 1 Optical properties of the irradiated phantoms. The concentration of IL refers to the fraction of solids in the solution, while the concentration of ink pertains to the fraction of the original product. Phantom #
Optical properties [mm 1]
Ink concentration [%]
IL concentration [%]
1 2 3 4
m ¼0.0064, ms0 ¼0.8 m ¼0.0073, ms0 ¼0.8 m ¼0.0096, ms0 ¼0.8 m ¼0.0192, ms0 ¼0.8
2 10 4 2.2 10 4 3 10 4 6 10 4
0.8 0.8 0.8 0.8
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particulates, the light’s polarization status changes. For this reason, first we measured the polarization of light that passed through the scattering and absorption medium of the static phantoms. We tested different types and widths of phantoms. The phantoms were prepared as described in Section 2 and four different phantoms that are presented in Table 1 were measured. The samples were prepared in order to mimic the optical properties of real biological tissues. Each sample was measured for varied widths of 1, 2, 4 and 5 mm. In Fig. 2 one can see representative results for the 4 different phantoms (from Table 1). The polarizers were positioned perpendicularly to one another, so that when no sample was inserted between them no light was collected by the camera and the polarization state (the normalized intensity along the direction of the output polarizer) remained 1 (marked as sample #0 in Fig. 2). When inserting the samples (1, 2, 4 and 5 mm wide phantoms with the characteristics described in Table 1), one could see a small reduction in the polarization state, but in all cases the polarization remained higher than 0.92. These results are in high agreements with previous reports [23,24]. 3.2. Polarization of real tissues
Fig. 1. Image (a) and optical diagram (b) of the experimental setup. A laser illuminates a sample after passing through the left polarizer and the transmitted light is imaged by a camera after passing through the right polarizer.
velocities, we used 1, 1.5 and 2 ml of tap water, purified water, PBS (pH¼7.4) and Dulbecco’s modified Eagle’s medium (including Calcium, Magnesium, Potassium and Sodium), which were injected to the tunnels by an engine bundled to different syringes, where the content of the syringe was injected to the sample during the same period of time (10 s). The scattering properties of the tap water, purified water and PBS were zero; the optical properties of Dulbecco’s modified Eagle’s medium were not measured directly but before starting the flow, the first control measurement was done including the sample and the medium without flow. We planned the above flow velocities (6–12 ml/s), volumes and tunnel diameters in order to achieve typical human blood flow velocities. The blood flow in the entire human circulation is about 5000 ml/min in an average sized adult at rest, but may be 5–6 times greater during heavy exercise [30]. The blood vessel diameters change from a few mm down to microns, so our rates and diameters are similar to the neck vein blood flow velocity [31]. By the term of polarization we refer in this research to the following ratio of measured intensities: P¼
Solid phantoms composed of ink and interlipid suspended in water may have bulk optical properties similar to those of a tissue (absorption and scattering coefficients, anisotropy of scattering, etc), but one may claim that they will not likely recapitulate tissue properties. The reason might be that the nature of the refractive index variation in the phantom is caused by discrete particulates, while tissue is a random continuum. In addition, it does not necessarily imply that the polarization properties of the phantom match those of the tissue [28]. In order to test this issue, measurements of real tissues (pieces of uncooked turkey meat) were performed and compared to phantom samples with similar optical properties [32]. The measurements were done for different widths of tissues and phantoms (1–4 mm). As can be seen from Fig. 3, the polarization properties in these tissues are identical to those measured in phantoms. Since scattering in static tissue structures rapidly depolarizes an incident beam, we have chosen to use samples whose polarization state remained high ( 40.9) without flow and only the flow depolarization takes effect. 3.3. Flow inside the phantoms Measurements of flow in phantoms were done as follows: phantoms with the same characteristics as were used for the
I0,0 I0,90 I0,0 I0,90
where Ia,b is the intensity measured when the first polarizer was positioned at an angle a and the second at an angle b. The captured videos of the transmitted light were stored as avi files and analyzed using the MATLAB (The MathWorks, Natick, MA) software. In particular, avi movies were translated to individual frames where each one of them contains all the image information, such as its time and exposure duration.
3. Experimental results 3.1. Static phantom As polarized light propagates through light-scattering media, such as biological tissues, microsphere solutions or an atmosphere with
Fig. 2. Polarization measured by the experimental setup for different phantoms without flow. Four different phantoms with 4 mm width are presented and their optical properties are described in Table 1. The polarization of all samples is larger than 0.92.
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Fig. 3. Polarization for phantoms (diamonds) and for a piece of turkey meat (square) with different widths. Note that both samples have a contrast close to one, similarly to what was obtained in Fig. 2.
static phantom measurements and with 5 mm width were used for the flow measurements. Two different samples were prepared: one sample with a single tunnel of 0.8128 mm in diameter and another sample with a single tunnel of 0.7112 mm in diameter. The tunnels were generated in the middle of the samples. Four types of liquids (tap water, purified water, PBS and Dulbecco’s modified Eagle’s medium) at flow rates of 0.1 ml/s, 0.15 ml/s and 0.2 ml/s were injected into the tunnels. The flows were in different directions relative to the excitation polarizer. We measured flow in the same direction as that of the polarizer excitation, in a perpendicular direction to the polarizer and at 451. The results for the polarization state obtained for the purified water are presented in Fig. 4. When the polarizer and the flow were in the same direction there was almost no decrease in the polarization (still higher than 0.9) detected for all flow rates (black lines). When the flow was in a perpendicular direction to the light, the polarization state decreased up to 0.4 (green lines). For different flow rates we got different integration durations that generated a reduction of the polarization; for a flow rate of 0.1 ml/s in the perpendicular direction the polarization was reduced after 2 s to 0.6 (full thin green line), while for 0.2 ml/s flow velocity in the perpendicular direction the polarization reduced after 2 s to its final value of 0.4 (full thick green line). When the flow was at 451, we got the polarization values between the last two cases (red lines). For the other three types of liquids (tap water, PBS and Dulbecco’s modified Eagle’s medium) the results were the same (data not shown). These results, dealing with the different orientation of the polarizer relative to the flow direction, match a mathematical relation of cos2 y between the intensity in the perpendicular direction and the relative angle, meaning that the flow will not influence the polarization when it is in the same direction as the incident light’s polarization. However, when there is an angle of 901 between the polarization and the flow direction, the depolarization effect will be significantly stronger and at 451 the polarization is reduced by half. While at 01 we measure P¼ 0.9, at 901 P¼0.4; hence DP¼P(y ¼01)—P(y ¼901)¼0.5 and P(y ¼ 451)¼0.65 is exactly DP cos2 451þ P(y ¼901).
4. Conclusions and discussions The presented paper tries to understand the possible role of polarization of light in phototherapy. The biological tissue is not static and contains blood vessels and capillaries, where liquid is
Fig. 4. Polarization for phantom with different flow conditions. Three different flow rates were measured for three different orientations of the flow-polarizer. The full thin lines present a flow velocity of 0.1 ml/s; the dashed lines present a velocity of 0.15 ml/s; the full thick lines present a velocity of 0.2 ml/s; the black lines present flow in the same direction of the polarizer; the red lines present flow at 451 to the polarizer; the green lines present flow perpendicular to the direction of the polarizer. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
flowing in many directions. Therefore, we measured the polarization of light propagating through a biological tissue versus the volumetric flow rate and the integration time as well as the flow direction. We found that the polarization of the laser is almost uninfluenced by light passing through a static tissue with a low reduced scattering coefficient of ms0 ¼0.8 mm 1 or when the flow direction is parallel to the polarization direction of the laser. However, it is partially lost when there is a flow of fluid through the tissue in a direction perpendicular to the laser’s polarization direction. No evidence for optical activity was found. It should be noted that in the current study the tissues that we used was fresh flash tissues, which did not mimic the actual in vivo situation where blood is flowing within the living tissue. The volumetric flow rate is directly correlated to the polarization loss. Higher flow rate produces lower polarization values. This may happen due to the flow itself as well as due to the movements of the scattering elements inside the tissues, caused by the flow. The multi-scattering phenomena reduced the polarization value. The fact that the blood vessels in the body do not range in one direction means that the laser beam necessarily hits the fluid flow through the tissue perpendicular to its polarization direction. It can thus be concluded that from the polarization aspect – based on the fact that linear polarization of laser radiation after passing fluid media may be lost, there are no additional benefits in using it as an excitation source without maintaining the excitation direction; for example it may simplify pulse oximetry instrumentation. Furthermore, the change in polarization as a flow rate indicator could be used as a diagnostic tool for congestive heart failure. It might also follow wound healing rates in diabetic patients.
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