Controlled light field concentration through turbid biological membrane for phototherapy.

Laser propagation through a turbid rat dura mater membrane is shown to be controllable with a wavefront modulation technique. The scattered light field can be refocused into a target area behind the rat dura mater membrane with a 110 times intensity enhancement using a spatial light modulator. The efficient laser intensity concentration system is demonstrated to imitate the phototherapy for human brain tumors. The power density in the target area is enhanced more than 200 times compared with the input power density on the dura mater membrane, thus allowing continued irradiation concentration to the deep lesion without damage to the dura mater. Multibeam inputs along different directions, or at different positions, can be guided to focus to the same spot behind the membrane, hence providing a similar gamma knife function in optical spectral range. Moreover, both the polarization and the phase of the input field can be recovered in the target area, allowing coherent field superposition in comparison with the linear intensity superposition for the gamma knife.


Introduction
Among successful surgery, radiotherapy, and chemotherapy technologies, phototherapy is regarded as a promising therapeutic technique as it has a number of distinct advantages including minimal invasiveness, lower systemic toxicity, selective tumor destruction and function preservation [1,2]. Typically, there are two types of phototherapy methods: photothermal therapy (PTT) and photodynamic therapy (PDT). PTT uses optical absorbing agents to effectively convert photon energy into heat to increase the local temperature near the tumor region [3,4], while PDT relies on cytotoxic singlet oxygen or reactive oxygen species from the photochemical reactions to induce tumor cell necrosis and/or apoptosis [5][6][7][8]. In any case, accumulation of photon energy in the target region is required while collateral damage to healthy tissues should be avoided [9,10].
A great deal of researches have been focused on the optical absorbing agents with high absorption in red or near-infrared light, since light can penetrate deeper into the tissue at these wavelengths [11,12]. However, optical scattering in bio-tissue is in general strong and light will be diffused before reaching tumors subjecting to phototherapy. The scattering becomes especially serious for visible and near ultraviolet light which is frequently used for PDT [3,13]. The optical scattering is usually caused by the nature of inhomogeneous structure distribution in a bio-system [14-16]. When a coherent light wave undergoes multiple scattering, the wavefront will be distorted and a volume speckle field is generated. Vellekoop et al. demonstrated a wavefront shaping technique which can focus the coherent light through disordered scattering media by using a spatial light modulator (SLM). By modifying the relative phase front of the incident beams, the fields can be made interfered constructively at a chosen point behind the scattering medium [17,18]. With a known structure as a reference, we demonstrate that the structures in the known vicinity can be recovered through thin turbid layers, and even the polarization of the input field can be restored by wavefront shaping [19,20]. In this article, focusing of light field through a turbid biological membrane (rat dura mater) is studied and the possibility of applying this technique to phototherapy for brain tumor is discussed. The experiment result shows a more than 200 times enhancement on power density in the target area compared with the input power density on the dura mater membrane, demonstrating the potential to improve the therapeutic efficacy of phototherapy while the effect of undesirable damage during laser delivery can be minimized. A multi-beam concentration system is proposed and presented for multi-angle stereotactic therapeutic scheme, hence providing a similar gamma knife function in optical spectral range.

Light field concentration through a scattering biological tissue with wavefront shaping technique
In this experiment, the scattering biological tissue is a rat dura mater. Dura mater is a kind of dense connective tissue adjoining the inner surface of the skull. It is also a kind of repair materials widely used in the clinic. Typically, the rat dura mater thickness changes from 0.1 to 0.3mm with age [23]. The sample is obtained from a two-month old adult rat. All the protocols have been approved by the Institutional Animal Care and Use Committee at Sun Yat-sen University in Guangzhou and are in compliance with the National Institutes of Health guidelines for the use of experimental animals. The dura mater was clamped between two pieces of glass sheets filled with phosphate buffered solution.
The schematic diagram for the experimental set-up is shown in Fig. 1. A collimated and expanded 632.8 nm He-Ne laser illuminates the SLM (Holoeye Pluto-VIS, binding 20x20 pixels as one unit). The incident beam is spatially shaped by the SLM for the compensation of the light scattering occurring in the bio-structure. A 3 times de-magnification telescopic system is used to image the SLM onto the surface of the scattering sample and provide necessary spatial resolution required for phase distribution. The focal lengths of the two lenses L1 and L2 are 30 cm and 10 cm respectively. The incident beam is set in an appropriate diameter to avoid the dura mater membrane being destroyed by the laser. In the experiment, genetic algorithm (GA) was used to search for the appropriate phase pattern to compensate the scattering of the dura mater scattering effect. A photo diode, with a pinhole of 50 micrometer diameter in the center, is used to measure the intensity at the target area. The level of the intensity serves as the feedback for GA. A MATLAB program is built to control the photo diode and SLM to find the optimal phase pattern through GA (Matlab Optimization Toolbox). A non-polarizing beam splitter placed between the biological tissue and the photo diode creates an image on the CCD camera for observation of the focus spot. Fig. 1 The system schematic of wavefront shaping and light concentration. A 632.8nm He-Ne laser beam is shaped by an SLM to penetrate through the dura mater membrane and focused to a spot. Figure 2(a) shows the intensity distribution transmitted through the dura mater without wavefront modulation. The incident light is scattered by the dura mater membrane and only a random speckle pattern can be detected. After the optimization process, the wavefront is optimized to compensate the diffusion process occurring in the dura mater membrane. As a result, the transmitted light is focused to the target area and a bright spot is generated, as shown in Fig. 2(b). Figure 2(c) shows the iteration process of the genetic algorithm. The intensity at the target area is enhanced by a factor of 110 after 700 iterations, and the target function tends to convergent. Under current conditions, it takes tens of minutes to finalize the 700 iterations of one optimization process. The optimal phase pattern calculated by the GA is shown in Fig. 2(d).
The transmitted field in the target, Em, is a linear combination of the fields coming from the N different segments of the modulator, written as [17]:  The intensity enhancement is defined as the ratio of the target intensity and the average background intensity of the speckle field without phase modulation. It is related to the number of controllable units on the SLM and to the state of the biological tissue. The intensity enhancement is of great importance to the therapeutic efficacy of phototherapy since regional tumor necrosis is positively correlated to the light dose in PDT [24]. As the energy concentration on the target, the dose of phototherapy agent could be reduced in comparison with the normal PDT and PTT. It is helpful for less toxic effect. Current PTT depends on absorbing agents to convert photon energy into heat. However, high power wavefront shaping concentration system shows the possibility to use the thermal effect of the concentrated light to do the photothermal therapy without absorbing agents.
To reduce the side effect on the surrounding normal tissue, the energy should be concentrated to the tumor region while the normal tissue should experience a minimum light intensity illumination. It is required that the incident surface, here the dura mater, should not be damaged by the input light field before the lesions are destroyed. The power density is measured at the target area and at the surface of dura mater respectively. The result shows a 200.7 times enhancement (Table 1). Thus, this experiment demonstrates the possibility to avoid dura mater damage while the tumor behind it is experiencing a higher light fluence while increase the laser power. The collection efficiency is defined as the ratio of intensity received in photo diode to the total input intensity on the dura mater. With this experiment configuration, the value is 2.35% ( Table 1). The collection efficiency is related to light dosimetry in phototherapy, which involves light fluence, exposure time etc. It shows the possibility to evaluate tumor cell necrosis and the efficacy of phototherapy.

Multi-beam concentration system
The thickness of human brain dura mater is typically 0.3-0.9 mm and the typical reduced scattering coefficient is about 16cm −1 at 632.8 nm [25,26]. We calculated the reduced scattering coefficient of the rat dura mater according to the method and parameters introduced in reference [25, 26], which is found to be about 10 cm −1 at 632.8nm. Hence, human dura mater scatters light more strongly, leading to the deterioration of wavefront optimizing performance. The total light transmission might be lower in human dura mater even with optimized phase pattern imposed with SLM. Then, a multi-beam light concentration phototherapy system is required for further light concentration in the stronger scattering system. As for a principle demonstration, only dual beam is discussed in this article. It is necessary to point out that this scheme is similar to a gamma knife which can concentrate dozens of gamma rays to a target area achieving single, high-dose external irradiation to the lesions [27] For dual-beam phototherapy scheme, the laser output from SLM is divided into two parts, as shown in Fig. 3(a). The two light beams are spatially separated before illuminating the dura mater at different positions. Using a similar optimization algorithm demonstrated in part 2, the light behind the dura mater tends to focus to the same target area in succession [ Fig. 3(b)] and generate two spots with intensity of I 1 = 0.224 a.u. and I 2 = 0.278 a.u. respectively . The polarization states at the two light spots are measured separately. It is found that the polarization states remain almost the same as the input after the optimization process. Our recent work showed that the polarization state through a strong scattering medium is actually controllable with different phase pattern in SLM [20]; hence the technique of multi-beam input is expected to be applicable to stronger scattering medium such as a human dura mater.
The two light fields may experience field superposition, which is related to the relative phase difference in between, as well as to the coherent property of the individual field. In the dual beam concentration experiment, one beam is maintained the calculated optimized phase, while a varying constant phase is added onto the other optimized beam. The intensity on the intersection area (target area) of the two beams shows a sinusoidal variation with the relative constant phase [as shown in Fig. 3(c)]. The sinusoidal variation of the total intensity with the compensated phase is a clear indication that the light through the bio-structure is experiencing field superposition rather than intensity superposition as occurred for gamma ray. As shown in Fig. 3(c), the total intensity of two optimized spots is 0.502 a.u [ Fig. 3(c) red line] when they experience intensity superposition. With the field superimposed, the maximum total intensity shows 1.7 times enhancement over an intensity superposition when they are in phase for a constructive interference [ Fig. 3(c) point A]. The picture on the right column of Fig. 3(b) shows the image of dual beam constructive interference. While the dual beam configuration works better than a single beam, it cannot reach the case of idea field superposition [as shown in Fig. 3(c) blue line]. Some reasons are accounted for why the maximum intensity is inferior to the theoretical one. Firstly, there is some of the phase modulation residue originated from the reflection of the liquid crystal pixel grids. Secondly, there is a mismatch between the pinhole diameter and the dimension of interference. And last but not least, scattering will generate tiny difference on the polarization states between two beams.
Considering multi-beam propagating onto the dura mater membrane at different positions, there will be a multi-field coherence at the target area and the target intensity follows the equation: Where M is the number of beams, An and Фn are the amplitude and the phase of the n th light beam respectively. If all the beams interfere constructively, the target intensity will enhance M 2 times than a single beam. In comparison with the intensity superposition of the non-coherent beam (such as the gamma ray), a more intensive concentration light field can be attained.

Discussion
By applying the wavefront modulation, we demonstrate that light can penetrate and focus through biological tissues. The most attractive advantages of using wavefront technique in phototherapy are penetrable localization and selectively light focusing. The selectively focusing in transverse direction and the penetrable localizing can be achieved by adjusting the feedback detector or the beam geometry.
The experiment of multi-beam concentration shows advantages of more precisely localization and higher power density in the target area over single beam system. The light focusing in the optical spectrum shows a higher energy concentration over gamma knife because of coherent superposition of the input fields. These features may lead to a reduced therapeutic exposure time with minimum damage to the biological structures of the dura mater.
In this experiment, a photo diode was placed behind the dura mater to provide feedback signal for the GA optimization process. In practice, a noninvasive detection system is desirable. In this case, fluorescence signal from fluorescent materials or intracellular autofluorescence in biological tissues can be monitored and used as the feedback [28,29]. Controlled light focusing can also be realized by using the ultrasonically encoded light or photo-acoustic signal induced by an absorber as feedback to guide the optimization [30, 31]. As the wavelength of ultrasonic wave and photo-acoustic wave are large compare to the inhomogeneity structure of the usual bio-system, they have higher transmittance through scattering tissues.
For the high power wavefront shaping concentration system, thermal effect may be accumulated in the target area. As indicated in reference [11], when cellular temperature exceeds 39°C, protein denaturation starts, and temperatures above 41°C temporary cell inactivation could last for several hours. "Severe" hyperthermia treatments (43-45°C) cause long term cell inactivation. Current PTT depends on absorbing agents to convert photon energy into heat. However, high power wavefront shaping concentration system shows the possibility to use the thermal effect of the concentrated light to do the phototherapy without phototherapy agent. The thermal effect can be monitored by using high sensitivity infrared thermal imager or infrared thermometer and the result can be used as feedback to the optimization process.
Another issue that needs attention is the duration of the procedure to achieve optimized wavefront modulation for optical energy concentration. Living biological tissues are always unstable and sometimes pulsatile. The wavefront should be optimized continuously and dynamically follow the changes in the biological system [21]. The current GA with a low refresh rate liquid crystal SLM is the most time consuming part in the optimization process. Cui reported a spatial frequency modulations technique that can provide a 400 milliseconds high speed for the entire wavefront determination using scanning mirrors and liquid crystal SLM [32]. Other spatial light modulators, based on deformable mirror devices, digital micromirror device and MEMS can provide a higher modulation speed than that obtainable with a liquid crystal SLM [33][34][35]. Other phase finding algorithm such as phase shifting method and coordinate descent optimization method can provide even higher speed [34, 35].

Conclusion
In conclusion, we show that light can be guided through turbid biological membrane and focused to a target point through wavefront modulation technique using an SLM. A dualbeam light concentration phototherapy system for rat dura mater membrane is demonstrated and the possibility of using multi-beam for phototherapy is discussed. Besides, this technique has potential applications in other subcutaneous lesions, intracorporal hydatoncus or tumors by coupling light with an optical fiber to deliver it to the target biological structures.