1. Near infrared spectroscopy system for dynamic and static measurements of oxygenation and blood volume changes in a liquid phantom Experimental setup We have used for our cwNIRS experiments an inexpensive continuous-wave DOT system [3] containing a spatially combined 2 laser diode sources RLT780-1000G (1W at 785 nm) and RLT83500G (0.5W at 830 nm) from Roithner Lasertechnik, a variable-gain photo detector OE-200-SI equipped with 1.2 mm active diameter Si photodiode and two lock-in amplifiers LIA-MV-150 from FEMTO Messtechnik GmbH, which can acquire 128 independent measurements in about 60 seconds through two time- division-multiplexed optic scheme with eight illumination fibers and eight detector fibers in the measuring head (optode). The beams of the two laser diodes are collinearly overlaid and coupled into one of multiple source fiber bundles using an optical coupler Patchcord-1m-1000/1035/1400 LOH HCS 0.37NA-FC-FC-PVC (RoMack Inc.) and four collimators two C230220P-B and two C220MP-B (ThorLabs Inc.). The serial activation of a given light source from the measuring head is possible through an optical demultiplexer DMUX (single-input, multiple-output switch) realized with a translation stage PI M505.2S2 (Mercury Inc.), which moves precisely in front of the illumination fibers, the collimator connected at the optical coupler of the two laser diode. The light transmitted through the target media (tissue phantom) is measured using a similarly serial approach, but through an optical multiplexer MUX (multiple-input, single-output switch) which moves precisely in front of the detecting fibers the collimator placed at the input of the photo preamplifier. Each source/detector fiber bundle is fixed in a given order and alignment, on a mechanical mount placed in front of the illuminating/receiving collimator of the optical switches DMUX and MUX respectively. A microprocessor-controlled stepper motor model M505.2S2 (20.000 counts/rev.) providing ultra-smooth, vibration free 0.1 μm minimum incremental motion, allows a relatively fast and precisely positioning of the collimator in front of the illumination/measuring fibers in a 50 mm travel range. All optical fiber are uniformly pressed on the liquid phantom glass wall (Figure 3). Principles of cwNIRS Measurement Device According to the modified Beer–Lambert law (MBLL), the optical density (OD) variation ΔOD(r,s;λ,t) (unitless quantity) at time t due to oxyhemoglobin (HbO 2 ) and deoxyhemoglobin (Hb) concentration changes (ΔCHbO 2, ΔCHb) [μM] is described as (1) where (r,s) is the detector and source position, λ the wavelength of the laser source, I(r,s;λ,t) the measured photon flux at time t, Io(r,s;λ) the initial photon flux, εHbO 2 (λ) [μM −1 mm −1 ] and εHb(λ)[μM −1 mm −1 ] are the extinction coefficients of the HbO 2 and Hb, DPF(r) is the unitless differential path length factor, and d(r)[mm] is the distance between the source and the detector at the position r, respectively [1,2]. Defining that OD λ the attenuation in intensity of light as a function of wavelength λ, this attenuation is the superposition of absorption (A λ ) and scattering (S λ ) of light with wavelength λ. For two chromophores contributions, the equation (1) can be rewritten as: (2) where λ indicate a particular wavelength. By measuring ΔOD λ at two wavelengths (λ 1 and λ 2 ) and using the known extinction coefficients of oxyhemoglobin (εHbO 2 ) and deoxyhemoglobin (εHb) at those wavelengths, we can then deter-mine their concentration changes: (3) (4) Assuming that concentration changes are both global and small and (5) the solution of the photon diffusion equation for a semi-infinite medium is: (6) where μ a,λ is the absorption coefficient and μ' s,λ is the reduced scattering coefficient at wavelength λ. This shows that ΔOD depends on tissue scattering, initial chromophore concentration, extinction coefficient and optode separation d. Blood chromophore information can be used to estimate blood volume and tissue oxygenation which are indications of hemodynamic activity. Different approaches can be used to implement NIRS such as time resolved, frequency domain and continuous wave techniques. Among these methods, continuous wave (cw) NIRS is the most practical one, where light with constant amplitude is injected to tissue and amplitude decay of the light intensity due to absorption is analyzed. Changes in light amplitude are used to calculate changes in concentrations of blood chromophores. Due to its practicality cwNIRS systems allow bedside monitoring of blood chromophores for extended periods. Since each chromophore has a specific extinction coefficient and differential pathlength factor, measurement with two wavelengths can be expressed simply by matrix, namely: (7) where Therefore the equation (7) provides a transformation from light output in blood chromophore concentrations. By using blood chromophore concentrations we define two parameters, namely, OXY = ΔCHbO 2 - ΔCHb (8) and BV = ΔCHbO 2 + ΔCHb (9) OXY and BV are estimates proportional to, oxygenation and blood volume changes in the tissue due to hemodynamic activation. R. Cernat, D. C. A. Dutu, S. Banita, M. Patachia, and D. C. Dumitras Department of Lasers, National Institute for Laser, Plasma and Radiation Physics, 409 Atomistilor St., PO Box MG-36, 077125 Bucharest, Romania e-mail: ramona.cernat@inflpr.roramona.cernat@inflpr.ro Abstract Optical measurements based on multiple source-detector positions on the tissue surface can reconstruct the internal distribution of the absorption coefficient (µ a ) and the reduced scattering coefficient (µ´ s ) in two or three dimensions based on the light transport model. Optical imaging using diffusing light offers potentials for a number of biomedical applications such as breast-cancer detection and tissue oxygenation mapping. However, these methods have been based on techniques in which the measured data are used directly to form the image, a process that may not take full advantage of the information contained in such data. The procedure of estimating blood chromophore concentrations by means of near infrared light is called Near Infrared Spectroscopy (NIRS). Blood chromophore information can be used to estimate blood volume and tissue oxygenation which are indications of hemodynamic activity. The chromophore distribution analysis through cwNIRS system can enhance the resolution and accuracy in various deep-tissue applications, including breast cancer imaging through diffuse optical tomography (DOT). Fig.1 An overview of the DOT and cwNIRS experimental setup. Fig.2 The measuring head (Optode) Fig. 4 The cylindrical beaker used for liquid tissue phantom Fig. 3 The two displays used for the two level software control cwNIRS Instrumentation We have developed a continuous-wave near infrared spectroscopy (NIRS) system using the hardware structure of the cwDOT system previously described, using its two spatially combined laser diode sources (at 785 nm and 830 nm operating wavelengths) and a measuring head (optode) with eight illumination fibers and eight detector fibers. This complex structure enable us to investigate the signal dependence on the distance between light source and receiver and the dynamic spectral measurements when the system uses eight special distributed light sources and eight receivers. A liquid phantom simulating the optical properties of the tissue was used in order to test the efficacy of the system. The basis of the phantom was formed by a scattering solution of intralipid (Lipofundin) and water with an overall reduced scattering coefficient of 0.8 mm -1 (785 nm). The solution was placed in a cylindrical beaker, a magnetic stirring rod was used to maintain the homogeneity during the experiment for the dynamic regime. Red blood cells obtained from healthy human blood were added to scattering solution to achieve a volume fraction of 1.5% and a total hemoglobin concentration of 30 μM. This is a typical value for normal physiological conditions with an assumption of 1.5% blood volume and 15% hematocrit. The hemoglobin saturation was measured to be 85%. In order to induce deoxygenation on the liquid phantom, 4 g of Bakers yeast was added to the solution. The temperature of the phantom was maintained during the deoxygenation process at 37°C to keep the yeast active. Calculation and parameters Absorption and reduced scattering coefficients used in the calculation of the DPF λ have the values given in the literature [4]. An assumption of 85% saturation and 150 μM total hemoglobin concentration results in the absorption and reduced scattering coefficients of Using these coefficients in equation (5), we obtain the DPF values for different distances d between the light source and receiver at the two specified wavelengths, as in Table 1. Table 1 These values are consistent with the measurements in the literature [4]. On the other hand, from the values given in the literature [5] we have: (10) By inserting into equation (7), the relationship between light intensity change and blood chromophore concentration is derived for different d values, as: d (mm) = 9.75 d (mm) = 19.13 (11) d (mm) = 27.75 d (mm)DPF 785 nm DPF 830 nm 9.754.0197874.037241 19.134.8341664.708042 27.75.1730994.975389 a b Fig.5 The experimental setup with the blood liquid phantom in the measuring head: a) the phantom color is bright red when the oxy-hemoglobin concentration is high and b) dark red when the hemoglobin concentration is high. Results A dynamic liquid phantom simulating the optical properties of the tissue was used in order to test the efficacy of the system. The solution was placed in a special cylindrical beaker designed in the lower part with an oxygen supply pipe. The oxygen pipe is closed by a porous glass wall which avoid the penetration of the liquid in the oxygen pipe line and improve the oxygen bubbles distribution in the liquid phantom volume (Figure 4). Deoxygenation and oxygenation of the hemoglobin was realized periodically by immersing a small yeast bag in the liquid phantom and delivering extra oxygen to the phantom from an oxygen tank, respectively. Oxygen supply was maintained until a steady state level of oxygenation was obtained. The signal evolution in the output of the two lock-in amplifier is proportional with the photon flux measured for each wavelength by the receiving photodetector placed at the distance d from the light source and describe the chromophore concentration during the oxygenation and deoxygenation processes in the blood phantom (figure 6). According to the equation (1), (3), (4), (8) and (9) using the known extinction coefficients of oxyhemoglobin (εHbO 2 ) and deoxyhemoglobin (εHb) (10) and the two input light intesities applied on the probe, we can compute the oxy and deoxy hemoglobin concentration (Figure 7) and OXY and BV (Figure 8). The same result is obtained also if the concentration is measured dynamically using the 8 pairs of sources and detectors of the measuring head and a software which drive the two optical multiplexors to connect periodically each adjacent source-detector pair SiRi in the measuring system (Figure 9). Conclusion The use of the NIRS algorithms with the DOT image reconstruction techniques improves the tissue characterization, because NIRS is the only method that can potentially measure hemodynamic metabolism and neuronal signals simultaneously. cwNIRS brings together some characteristics that are usually noninvasive and provide good spatio-temporal resolution. The main advantage of the functional NIRS (fNIRS) is the ability to measure a wide range of functional contrasts such as oxy-hemoglobin, deoxy-hemoglobin and total hemoglobin directly with very high temporal resolution. Based on tumor hemoglobin and blood volume fraction in the pathologic zone measured by cwNIRS the DOT can precisely separate the normal structure from a malignant one. References 1.J. Heiskala, K. Kotilahti and I. Nissilä, "An application of perturbation Monte Carlo in Optical Tomography", Proceedings of the 2005 IEEE Engineering in Medicine and Biology 27th Annual Conference, Shanghai, China, September 1-4 (2005). 2.J. Chul Ye, S. Tak, K. Eun Jang, J. Jung, J. Jang, "Statistical parametric mapping for near-infrared spectroscopy", Neuroimage 44, 428 – 447 (2009). 3.D.C.A. Dutu, D.C. Dumitras, C. Matei, A. M. Magureanu, M. Patachia, S. Miclos, D. Savastru, X. Liang, H. Jiang, and N. Iftimia, "Design and parametric evaluation of a continuous-wave diffuse optical tomography system", ICPEPA ’09 Sapporo, Japan (2009) 4.A.Bozkurt, A. Rosen, H. Rosen and B. Onaral "A portable near infrared spectroscopy system for bedside monitoring of newborn brain“, BioMedical Engineering OnLine (2005). 5.Scott Prahl, “Tabulated Molar Extinction Coefficient for Hemoglobin in Water” (prahl@ece.ogi.edu). Magnetic stirring rod Oxygen pipe Advanced Laser Technologies ALT’10 Fig. 6 The voltage measured at the output of the two lock-in amplifiers ( the signal measured on the wavelength 785 nm and 830 nm respectively is decoded by the specific modulation frequencies) Fig. 7 Time evolution of the oxy and deoxy hemoglobin concentration in the tissue phantom under hemodynamic activation through oxygen flow and yeast adding. Fig. 8 The time evolution of the OXY and BV under hemodynamic activation through oxygen flow and yeast adding. The oxygenation and deoxygenation rate are dependent by the blood concentration oxygen flow rate and yeast quantity. Fig. 9 OXY and BV temporal evolution under hemodynamic activation using the dynamic data acquisition of the DOT system.