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IEEE TRANSACTIONS ON MICROWAVE THEORY AND TECHNIQUES, VOL. 52, NO. 8, AUGUST 2004 2057 High Peak SAR Exposure Unit With Tight Exposure and Environmental Control for In Vitro Experiments at 1800 MHz Jürgen Schuderer, Theodoros Samaras, Member, IEEE, Walter Oesch, Denis Spät, and Niels Kuster, Member, IEEE Abstract—The objective of this study was to develop, optimize, and characterize a flexible and reliable unit for standardized and well-controlled electromagnetic-field exposure of cells in vitro. The technical requirements were high peak and time-averaged exposure of the cells with a minimal temperature rise for the cell cultures, flexible modulation schemes, high uniformity, and low variability of exposure, as well as support of blinded protocols. The developed setup is based on two R18 waveguides resonant at 1800 MHz and operated with a computer-controlled signal and monitoring unit. The cells can be exposed in 35-mm Petri dishes either cultivated as monolayers or in suspension. For cell monolayers, the system provides an efficiency for the specific absorption rate (SAR) of 50 (W/kg)/W input power, a nonuniformity of the SAR distribution of 30%, SAR variability of 6%, and a temperature rise of 0.03 C/(W/kg) average SAR. For cell suspensions and provided that the cells are not in the meniscus area, a SAR efficiency of 10 (W/kg)/W, nonuniformity of 40%, SAR variability of 17%, and a temperature rise of 0.13 C/(W/kg) is achieved. The numerical dosimetry for the field and temperature distributions within the Petri dishes was verified using -field and temperature probes. The temperature analysis has shown that the possibility of localized “hot spots” can be excluded. Index Terms—Dosimetry, absorption rate (SAR). exposure setup, RF, specific I. INTRODUCTION I N VITRO studies are important for the detection of the biological effects of RF electromagnetic field (EMF) exposures, e.g., as emitted by mobile phones. They can be used to identify basic mechanisms and to analyze functional and structural changes in living cells. The objective of this study was to develop a standard in vitro exposure system operating in the digital communication system (DCS) frequency band of the global system for mobile communications (GSM) at 1800 MHz. The setup shall be used for various diverse study endpoints by a multitude of laboratories working within the European research program. Therefore, the setup should fit inside commercial incubators and needs to provide high flex- Manuscript received October 15, 2003; revised March 23, 2004. This work was supported by the Swiss Agency of Education and Science, by the Mobile Manufacturers Forum, by the GSM Association, and by Sunrise TDC. J. Schuderer, W. Oesch, D. Spät, and N. Kuster are with the Foundation for Research on Information Technologies in Society, Integrated Systems Laboratory, Swiss Federal Institute of Technology, CH-8092 Zürich, Switzerland (e-mail: [email protected]). T. Samaras is with the Department of Physics, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece. Digital Object Identifier 10.1109/TMTT.2004.832009 ibility with respect to different exposure schemes and cell culturing conditions. Among the anticipated conditions, cells might by cultivated as a monolayer on an artificial substratum, e.g., the plastic bottom of a Petri dish, or might be grown in suspension, such as blood cells in plasma. In particular, the following requirements for the RF exposure system posed by the European research program consortia were derived from [7]. • SAR Requirements. 1) The temperature rise of the cells as a result of the exposure should be insignificant (i.e., 0.1 C) for levels of the specific absorption rate (SAR) as high as the International Commission for Non-Ionizing Radiation Protection (ICNIRP) limit for local exposures set for the general population, i.e., SAR of 2 W/kg [5]. 2) The setup needs to provide high SAR efficiency since exposures at this level with signals of high crest factors1 demand high peak SAR exposures (e.g., 150 W/kg for the discontinuous transmission mode (DTX) of GSM). 3) The nonuniformity of the SAR over all cells should be less than 30% (minimal sample area for cell monolayers: 50 cm , minimal sample volume for cell suspensions: 10 mL). 4) The combined uncertainties of the SAR assessment and possible exposure variability due to drifts and other variations between different experiments should not dominate the SAR nonuniformity, i.e., should be less than 30%. 5) The power isolation between exposure and sham must be more than 30 dB. • Signal Requirements. A flexible signal unit is required to enable complex modulation such as: 1) continuous wave; 2) pulse or sinusoidal modulation at any frequency and repetition rate; 3) GSM signals simulating: i) the basic GSM mode (basic) active during talking into the phone, ii) the DTX mode active while listening, iii) conversation covering temporal changes between basic and DTX, and iv) network environment covering environmental power control and handovers; 4) other time division multiple access (TDMA) signals such as digital advanced mobile phone service (DAMPS), digital enhanced cordless telephone (DECT), personal handyphone system (PHS), etc. Additionally, intermittent exposure protocols with intermittence cycles from seconds to hours should be applicable. • Controlling Requirements. Identical environmental parameters for exposure and sham must be ensured, i.e., both 1Crest factor 0018-9480/04$20.00 © 2004 IEEE = ratio between the peak and average SAR. 2058 IEEE TRANSACTIONS ON MICROWAVE THEORY AND TECHNIQUES, VOL. 52, NO. 8, AUGUST 2004 systems should be kept in the same incubator and temperature differences between exposure and sham should be less than 0.1 C. Blinded protocols should be applicable, and the exposure and environmental conditions must be continuously monitored. The setup should be capable of self-detecting malfunctions. • Requirements for Dosimetry. The dosimetry should cover: 1) detailed numerical evaluation of the SAR distribution; 2) experimental verification by dosimetric measurements; 3) uncertainty and variability analysis for the SAR; and 4) evaluation of the temperature rise during exposure. Different setups have been used in the past for the exposure of cell cultures at the 800- and 900-MHz cellular frequency bands: Transverse electromagnetic (TEM) cells (e.g., [11], [6], [13]), RF chambers [9], radial transmission lines (RTLs) [10] and wire-patch systems [8]. Rectangular waveguides were employed in [17] for the DCS frequency band, and cylindrical waveguides were applied in [3] for personal communication services (PCS). Schönborn et al. [16] have qualitatively compared the performance of the TEM cell, RF chamber, RTL, wire-patch cell, and waveguide. For setups operating in the 1800-MHz region, the following conclusions can be drawn with respect to the formulated requirements, which are: 1) the RF chamber in -polarization cannot fulfill the requirements for SAR uniformity, SAR efficiency, and small size to fit inside an incubator. 2) The RTL setup provides good performance for studies with large sample volumes; however, it must be excluded for this study due to high costs and efforts to provide the required peak SAR and environmental control. 3) The waveguide setup operated at a cavity resonance can be expected to fulfill the requirements. It should provide: i) high SAR efficiency due to resonant operation; ii) small temperature rise for cell monolayers (low SAR for the whole medium at high SAR for the monolayer); iii) good uniformity of SAR when the Petri dishes are exposed in -polarization; and iv) good environmental control because exposure and sham chambers can be placed in the same incubator. Consequently, the developed setup is based on waveguide cavities that were optimized for cell monolayer exposures. In the course of the research programs, however, additional biological experiments have been added, requesting the exposure of cells cultivated in suspensions. For that purpose, a new configuration was developed enabling the exposure of cells in suspension with reasonable uniformity. Fig. 1. Side view of geometry and functional parts of the exposure system. The configuration for the cell monolayer is shown (all dimensions in millimeters). Inner dimensions of the R18 waveguides: 64.8 129.6 425 mm (height width length). 2 2 Fig. 2. 2 2 Exposure chambers with removed end-short plate. carrier frequency, which approximates exposure from mobile communication systems like DCS (uplink: 1710–1785 MHz; downlink: 1805–1880 MHz), PCS (uplink: 1850–1910 MHz; downlink: 1930–1990 MHz), and universal mobile telecommunication system (UMTS, uplink: 1920–1980 MHz; downlink: 2110–2170 MHz). B. Waveguide Cavity II. DESIGN OF THE SETUP Figs. 1 and 2 show the mechanical design of the realized exposure system. The concept is derived from [17]. Novel features have been developed and implemented to meet the requirements: A. Frequency R18 instead size of length: waveguides (cross section: 129.6 mm 64.8 mm) of the R16 waveguides [17] were used to reduce the the entire setup (height: 450 mm, width: 200 mm, 500 mm), but still enable the exposure of cells at a The waveguide and coupler were optimized to achieve a resonance with minimal field disturbance inside the waveguide cavity. This was achieved by adjusting the length for a resonator mode at 1800 MHz. A flat loop coupler (Fig. 3) on one end of the waveguide and an end-short plate on the other end were gold plated to ensure good RF contacts. The short plates were equipped with quick-mounting fasteners in order to allow access to the cavities. The loop coupler has the comparable advantage over the monopole -field coupler in that it reduces the extension of the evanescent mode region and, therefore, enables utilization of a larger proportion of the waveguide or a reduction of the length. SCHUDERER et al.: HIGH PEAK SAR EXPOSURE UNIT WITH TIGHT EXPOSURE AND ENVIRONMENTAL CONTROL FOR IN VITRO EXPERIMENTS 2059 an entire cultivation area of 60 cm are exposed to a uniform SAR distribution. D. Exposure of Cell Suspension Fig. 3. Side view of the loop coupler used for excitation of the waveguide cavities. Fig. 4. Petri dish holders for cell monolayer and cell suspension exposure. Dishes are placed inside the H -field maxima for monolayer and inside the E -field maxima for cell suspension cultures. TABLE I POSITION OF THE PETRI DISHES INSIDE THE R18 WAVEGUIDES The cavity loading is not significantly affected by the insertion of the Petri dishes ( -factor unloaded cavity 4000 versus -factor loaded cavity 1500). The 3-dB resonance bandwidth of the loaded cavity depends on the amount of cell medium in the Petri dishes and is in the order of 1–2 MHz, which is sufficient for all TDMA signals. The exact resonance frequency is determined prior to exposure by a frequency sweep for maximum field strength at the monopole field sensor. The narrow-band resonant design increased the SAR efficiency by a factor of 30 compared to [17], i.e., 50 (W/kg)/W input for 3 mL per dish. For cells in suspension, the efficiency is 10-(W/kg)/W input for 3.1 mL per dish. C. Exposure of Cell Monolayer Six 35-mm-diameter Petri dishes (effective inner diameter: 33 mm) are placed in the -field maxima of the standing waves (two dishes per maximum). A dish holder (Fig. 4) and distance keeper ensure the correct placement inside the waveguides and minimize spatial variability with respect to the incident fields (position accuracy for Petri dishes: 2 mm). The distances of the dishes to the short are given in Table I. Cell monolayers with Cell suspensions are exposed in the -field maxima of the resonator because a pure -field coupling is expected to result in a uniform SAR for a thin and flat dielectric (as for the suspended cell medium in the Petri dish). Four 35-mm Petri dishes, providing a sample volume of 12.4 mL are used (for positioning, refer to Table I). Due to stray fields in the proximity of the Petri dishes, the -field maximum at the field sensor location was not used for loading. Since strong coupling at the sides of the dishes is present, uniformity of the SAR is increased by the following loading procedure (Fig. 4): 35-mm-diameter dishes filled with 3.1 mL (liquid height: 3 mm) of medium are placed inside 60-mm-diameter Petri dishes (effective inner diameter: 54 mm). The resulting area between the dishes is filled with 4.9 mL of distilled water (maximum water volume to ensure no contact between water and Petri dish cover). A similar method was used in [8]. As for monolayer exposure, accurate positioning of the dishes ( 2 mm) is achieved by using a dish holder and distance keeper. E. Exposure Control Input power measurements provide poor accuracy for resonant structures and have been replaced by actual field measurements inside the waveguide. Since the cavity loading is low, measurement of the - or -field at one location only is sufficient to assess the incident exposure of all Petri dishes. The optimal solution would be a loop antenna at the short, since here, the location of the -field maximum is not dependent upon the wavelength. To avoid damage during loading of the dishes, a mechanically protected monopole antenna at the location of the first -field maximum before the short was preferred. Its length of 2.5 mm was optimized to cover a dynamic range of 50–10 000 V/m, 0.1–20 A/m when directly connected to a Schottky-diode detector (ACSP-2663NZC15, Advanced Control Components, Eatontown, NJ). The monopole and diode detecter were calibrated against an H3DV3 -field probe (SPEAG, Zürich, Switzerland) positioned at the location of an -field maximum. A low-pass filter is applied at the dc output of the diode to suppress noise. F. Signal Generation A fully computer-controlled signal unit was realized (Fig. 5). It is based on an RF signal generator (SML02, Rhode & Schwarz, Ittigen, Switzerland), an arbitrary function generator (33120A, Agilent Technol., Palo Alto, CA), a 5-W power amplifier (LS Electronik AB, Spanga, Sweden), a self-built radio frame generator and a data logger (34970A, Agilent Technol.). The general-purpose interface bus (GPIB) is used for software communication with the devices. Modulation can be applied in the following three pathways. 1) Amplitude modulation (AM) of the RF generator by the arbitrary function generator: any signal with a waveform length of 16 000 points, an amplitude resolution of 12 bits, and a frequency of less than 15 MHz can be used. 2060 IEEE TRANSACTIONS ON MICROWAVE THEORY AND TECHNIQUES, VOL. 52, NO. 8, AUGUST 2004 TABLE II MATERIAL PARAMETERS USED IN THE SIMULATION (" : RELATIVE PERMITTIVITY, : ELECTRIC CONDUCTIVITY, c: SPECIFIC HEAT CAPACITY, k: THERMAL CONDUCTIVITY, : MASS DENSITY) Fig. 5. Signal generation and monitoring unit (H: H -field, T: temperature, I : fan driving current, DL: data logger, PC: personal computer). 2) Software control of the output power of the RF generator: allows arbitrary field on/off intermittency and arbitrary power variations. 3) Blanking of the amplifier with the radio frame generator: is used to achieve idle frames; temporal changes between different radio frame structures can be realized. The data logger is used for the collection of all sensor signals and for the generation of digital control signals, e.g., for switching the microwave relay (SR-2MIN-D, RLC Electronics, Mount Kisco, NY). The measured field values are used for feedback regulation of the output power of the RF generator. In this way, amplifier drift and variations are compensated. G. GSM Signals The described concept is used for GSM modulation in the following way. The GSM burst, defined according to [2], is stored at the function generator and is applied to the AM modulation input of the RF generator. The frame structures of the basic crest factor and DTX crest factor modes are stored on the radio frame generator. Switches between both frame structures are software controlled and carried out by the data logger. Software regulation of the output power of the RF generator according to statistical functions is used to simulate the environmental events of a GSM phone conversation like channel fading, handovers, etc. The digital Gaussian mean shiftkeying (GMSK) modulation of the GSM signal is not applied since it has not been considered of significance for possible bio-responses. However, a vector RF generator can be easily integrated. H. Environmental Control Good environmental control is achieved by operating both waveguides inside the same incubator and enforcing rapid air exchange by fans (612NGHH, Papst, St. Georgen, Germany: air flow 56 m /h). However, it is not sufficient to place the waveguides next to each other since incubators can have a significant temperature gradient of several tenths of a degree. The requirement of less than 0.1 C difference between two unexposed waveguides is, therefore, only possible if the air used for atmospheric exchange for both waveguides enters from the same location within the incubator, e.g., by arranging the air inlets close to each other (see Fig. 1). I. Quality Control Quality control of the experiments is ensured by monitoring the exposure and environment, self-detection of malfunctions, and by blind protocols as follows. • Monitoring: The sensor signals ( -field, air temperature, fan driving currents) are continuously recorded by the data logger with a sampling rate of 0.1 Hz. All experimental data (settings, software commands, sensor signals) are stored in a file on the PC. • Blind Protocols: Blind study design is realized by randomly switching the microwave relay prior to exposure. Data files, including the information on which of the waveguides was exposed, are encoded. Decoding can be carried out by a dedicated program after biological evaluation. • Self-Detection of Malfunctions: The controlling and monitoring software is able to self-detect malfunctions and responds with warnings or shut-down if required (tracing and handling of 60 errors). A watchdog for PC shutdown was realized that does not allow any exposure without software control. III. METHODS A. Numerical Methods New dosimetric methods compared to [17] were applied: 1) Numerical Modeling: Field, SAR, and temperature distributions were characterized with a full three-dimensional (3-D) electrothermal finite-difference time-domain (FDTD) analysis using the simulation platform SEMCAD (SPEAG). The waveguide geometry was simulated with all dielectric materials, i.e., supporting plastic parts, Petri dishes, cell medium [Dulbecco’s modified eagle’s medium (DMEM)], and distilled water. The corresponding material parameters are summarized in Table II. As demonstrated in [18], reliable data can only be obtained if the meniscus profiles at the solid/liquid boundaries are modeled accurately. Therefore, all simulations have been performed with meniscus models based on the profile functions given in [18]. Four different medium volumes in the range from 2.2 to 4.9 mL (2–5-mm liquid height) were simulated for cell monolayer exposure. For cell suspensions, only one configuration with 3.1-mL DMEM and 4.9-mL distilled water was evaluated. A graded mesh with voxel sizes between 0.3–5 mm was applied for the discretization of the numerical model. A waveguide port was used for excitation SCHUDERER et al.: HIGH PEAK SAR EXPOSURE UNIT WITH TIGHT EXPOSURE AND ENVIRONMENTAL CONTROL FOR IN VITRO EXPERIMENTS 2061 TABLE III HEAT-TRANSFER COEFFICIENTS FOR THERMAL BOUNDARIES (for the assessment of SAR uncertainties, the loop coupler has also been simulated). Only a single total reflection at the short was simulated (results in the same standing-wave field distribution as for the resonator); the open free-space region at the source was terminated with absorbing boundaries. SAR -field value at a reference values were normalized to the position ( -field maximum at the short). 2) Numerical Evaluation: • Cell Monolayer: The cell monolayer is numerically represented as the interface between the FDTD voxels of the cell medium and Petri dish. Since fields at voxel edges cannot be directly derived from the FDTD implementation of SEMCAD, extrapolation of the SAR was used to assess an average value for the monolayer: the SAR values of all horizontal voxel layers within the cell media were evaluated, leading to an average SAR at a vertical distance , which is the distance between the voxel center and monolayer. Second-order polynomial functions were applied to . extrapolate the data to • Cell Suspension: SAR values were evaluated for the entire medium and for the medium excluding the meniscus (useful value for cells that are not in the meniscus). FDTD corner voxels at curved Petri dish/media interfaces were not considered for the evaluation because they are sensitive to numerical staircasing artifacts. The nonuniformity of the SAR distribution was quantified by the standard deviation of the SAR distribution and is expressed in relation to the average value. The lowest voxel layer of all six dishes was evaluated to derive the monolayer standard deviations, and all voxels representing the medium were used for suspension. The -field was additionally evaluated with the same methods in order to calculate the field impedance. 3) Thermal Simulation: A thermal FDTD analysis was performed for a single Petri dish model only (simulation of the entire setup geometry exhausts the computational resources). This model was analyzed for exposure inside a standing wave -field maximum for cell monolayers and -field maximum for cell suspensions. The standing wave was generated by two incident plane waves. This exposure configuration results in a similar SAR distribution as for the waveguide. Heat transfer due to conduction, radiation, natural convection, and forced convection was considered by solving the heat diffusion equation on the FDTD grid and by applying the appropriate thermal boundary conditions [15]. Combined heat transfer coefficients for convection and radiation were used to simulate the boundaries [1]. The coefficients were derived from a flat plate approximation of the cell medium [4]. The values of the heat transfer parameters are given in Table III. B. Experimental Methods The DASY3 near-field scanner (SPEAG) equipped with dosimetric field and temperature probes was used for field verification and for the determination of the temperature rise in the medium. Three-axis - and -field probes (SPEAG, EF3DV2, H3DV6) have been used to determine the incident field distribution in the vicinity of the Petri dishes. 1) SAR Verification: Different methods for SAR verification were applied as follows. • Monolayer Exposure: For cell monolayer exposure, a 1-mm-diameter -field probe [14] was used to map a vertical line in the center of the cell medium (4.9-mL DMEM). • Suspension Exposure: For suspension exposure, only single point temperature measurements were evaluated -field measurements were because the dosimetric considerably distorted by the strong incident -field parallel to the probe, leading to boundary effects and immersion depth errors [19]. The method of positioning the temperature probe (see below) did not allow the mapping of several points in the medium. During the SAR measurement, the fans were not operating. The first 50 s of exposure were evaluated by linear regression in order to derive the local SAR (via dT/dt). The temperature increase during this period was only 0.1 C; however, it was still enough for accurate evaluation because of the low noise level 0.005 C of the temperature probe (SPEAG, T1V3 thermistor). The 50-s evaluation period was assumed to be free of possible artifacts caused by thermal diffusion because SAR gradients in the vicinity of the sensor are very low and because the double layer of plastic provides good heat isolation toward the environment. 2) Temperature Rise: For the assessment of the temperature rise for the cells, measurements were performed within a 37 C, 95% humidity incubator environment. The probe was fixed inside the medium, 1 mm above the dish bottom. For the suspension geometry, the probe was carefully oriented perpendicular to the incident and induced -fields, which minimizes RF pick-up at the sensor leads. Additionally, a second probe was used with its leads guided in parallel to the first one, but placed below the Petri dish in the air. In this way, possible 2062 IEEE TRANSACTIONS ON MICROWAVE THEORY AND TECHNIQUES, VOL. 52, NO. 8, AUGUST 2004 Fig. 6. Simulated SAR distribution for monolayer and suspension exposure at 1800 MHz. Data is shown: (a) in a vertical plane in the center of the Petri dish, (b) in the lowest voxel layer (z 0:15 mm), and (c) in a histogram. = TABLE IV DOSIMETRIC DATA FOR THE AVERAGE SAR, NONUNIFORMITY OF THE SAR DISTRIBUTION, AVERAGE IMPEDANCE, TEMPERATURE RISE, AND THE CHARACTERISTIC TEMPERATURE RISE-TIME CONSTANTS FOR MONOLAYER (Ml.) AND SUSPENSION (Su.) EXPOSURES RF pick-up is similar for both probes and is compensated by differential temperature evaluation. No such artifacts are present for the monolayer configuration because the probe was located in an incident -field minimum. Measurements were performed at SAR levels of approximately 50 W/kg (monolayer) and 9 W/kg (suspension), resulting in an increase of approximately 1 C. The values were linearly scaled down with the SAR and normalized to 1-W/kg average SAR. The data for the temperature response of the medium was used for the determination of the maximum temperature rise at steady state and for the determination of the characteristic temperature rise-time constants. The latter allows assessment of the temperature time course for short periods or intermittent exposures. IV. RESULTS A. SAR Characterization Fig. 6(a)–(c) shows the SAR distribution for monolayer and suspension exposures in a vertical cut through the dish center, for the lowest voxel layer in the medium (monolayer only), and within a histogram, respectively. The internal - and -field are perpendicular to each other and polarized in the plane of the monolayer for the monolayer exposure. In contrast, the internal SCHUDERER et al.: HIGH PEAK SAR EXPOSURE UNIT WITH TIGHT EXPOSURE AND ENVIRONMENTAL CONTROL FOR IN VITRO EXPERIMENTS 2063 Fig. 7. Simulated SAR and steady-state temperature distributions (start temperature: 37 C, forced air-cooling active). The steady state is reached after approximately 15 min for monolayer and 30 min for suspension exposure. The location of the temperature probe used for the experimental assessment is indicated in this figure. -field is polarized perpendicular, and the -field is polarized parallel to the medium surface for cell suspensions. Statistical data for the exposure is given in Table IV: results for the average SAR values, nonuniformity of the SAR distribution, average field impedance, temperature rise, and thermal response time constants are reported. The exposure is strongly dependent upon the medium volume for cell monolayers and is characterized by high vertical SAR gradients. If cells are not in the meniscus area, an evaluation of SAR in the medium excluding the meniscus is appropriate and leads to much lower nonuniformity of SAR for cell suspensions (40% versus 117%). The field impedance for monolayer exposure is by a factor of five lower than for suspension. B. Temperature Rise A much lower temperature rise is present for monolayer exposure compared to suspension [3.1 mL: 0.022 versus 0.13 C/(W/kg)]. Fig. 7 shows the simulated SAR and steady-state temperature distributions for a 1-W/kg exposure (single Petri dish model). Temperature is uniformly distributed without localized temperature “hot spots” for both cell culture configurations. For cell monolayers, the location with the highest temperature rise is the center of the dish (which was also experimentally assessed). The probe was not placed within the temperature maximum during the measurements in cell suspension. Therefore, the simulated maximum temperature rise in the meniscus, which is 20% higher, is additionally given in Table IV. Fig. 8 shows the measured time response curves for a 51-W/kg monolayer and 9-W/kg suspension exposure. Fig. 8. Measured temperature rises during RF exposure for cell monolayer and suspension (3.1-mL cell medium, forced air-cooling active). The temperature probe was positioned inside the medium, 1 mm above the Petri dish bottom, for both configurations. The start temperature was 37 C. Average SAR values of 51 and 9 W/kg were present for the cell monolayer and cell suspension (without meniscus), respectively. C. SAR Verification, Dosimetric Uncertainty, and Variability The measured SAR distribution for cell monolayers is compared to simulations in Fig. 9. Measurement data is plotted for the three upper dishes, as well as for one lower dish. An average difference between simulation and measurement of 15% was found. For the cell suspension configuration, this difference for the single-point evaluation was 22%. 2064 IEEE TRANSACTIONS ON MICROWAVE THEORY AND TECHNIQUES, VOL. 52, NO. 8, AUGUST 2004 Fig. 9. SAR values as a function of the distance to the dish bottom for cell monolayer exposure. The corresponding slots are indicated in Fig. 1. TABLE V UNCERTAINTY ANALYSIS FOR THE SAR ASSESSMENT OF MONOLAYER (Ml.) AND SUSPENSION (Su.) EXPOSURES. ESTIMATED STANDARD DEVIATIONS WERE DERIVED AND ARE COMPARED TO THE DIFFERENCE BETWEEN MEASUREMENT AND SIMULATION These results must be discussed together with the uncertainties of the SAR assessment, as summarized in Table V. Uncertainty was evaluated according to the methodology of [12]. Normal or rectangular error distributions were assumed in order to derive an estimated standard deviation for each investigated uncertainty contribution. Combined uncertainties of 20% and 21% are specified for cell monolayer and suspension SAR assessment, compared to the average differences of 15% and 22%, as derived above. Since the difference is in the range of the uncertainties, the measured results verify the numerical assessment and support the reliability of the dosimetry. An analysis of SAR variability is provided in Table VI. A combined relative variability of 5.1% and 17% in relation to 23%–30% and 40% SAR nonuniformity is derived for monolayer and suspension exposures, respectively. V. DISCUSSION A. SAR Efficiency and Uniformity Both setups provide an excellent SAR efficiency of 50 and 10 (W/kg)/W for monolayer and suspension exposures, respectively, which is a clear advantage compared to other exposure systems. An inexpensive 5-W power amplifier is thus sufficient to achieve monolayer SAR values 200 W/kg. Signals with high crest factors such as the GSM DTX mode can be applied at the ICNIRP limit of 2 W/kg. However, the resonant operation also leads to some disadvantages: The carrier frequency is not fixed, but depends on the loading volume of the Petri dishes (1% difference for 2- and 5-mL medium volume). Secondly, amplitude and phase distortions can occur due to the restricted resonance bandwidth of 1–2 MHz. This problem is of no concern for the applied amplitude-modulated carrier signal with sidebands as multiples of only 217 Hz. Furthermore, only low distortions are present for a GMSK modulated GSM signal since its signal bandwidth of 200 kHz is still 5–10 times less the resonator bandwidth. However, the application of UMTS signals, which have a bandwidth of 5 MHz, is not possible under resonant conditions. For this case, a broad-band coupler, as applied in [17], should be used. The required uniformity of SAR with deviations 30% is only achieved for cell monolayer exposure and is in the same order of magnitude as for other exposure systems in the literature. The suspension configuration exceeds the requirement of a value up to 40% (without meniscus evaluation). If cells do not settle from the meniscus, a high nonuniformity of 117% is present (Table IV). Therefore, the sedimentation behavior of the cells must be analyzed carefully, and exposure should not be started until cells have settled from the meniscus. The provided sample area/volume with uniform exposure of 60 cm for cell monolayer and 12.4 mL for cell suspension is not superior compared to other systems. However, it was chosen in order to keep the dimensions of the setup small. For studies requiring a higher sample volume, usage of other setups such as the RTL should cm ). be considered (sample area B. Dosimetric Uncertainty The dosimetry led to uncertainties for the SAR assessment of approximately 20% with the highest contributions resulting from the measurement of the dielectric parameters and the SCHUDERER et al.: HIGH PEAK SAR EXPOSURE UNIT WITH TIGHT EXPOSURE AND ENVIRONMENTAL CONTROL FOR IN VITRO EXPERIMENTS 2065 TABLE VI VARIABILITY ANALYSIS FOR MONOLAYER (Ml.) AND SUSPENSION (Su.) EXPOSURES. ESTIMATED STANDARD DEVIATIONS WERE DERIVED AND ARE COMPARED TO THE NONUNIFORMITY OF THE SAR DISTRIBUTION calibration of the monopole sensor (Table V). Although no detailed uncertainty analysis is provided for other setups in the literature, the exposure characterization reported in this manuscript should provide a high level of accuracy compared to others because: 1) meniscus models for the cell media were used; 2) incident field instead of power monitoring is applied; and 3) simulations were successfully verified by measurements. Additionally, for the first time, a full 3-D thermal analysis was performed to assess the temperature rise in the Petri dishes. This method allows clear interpretation of the biological results with respect to thermal or nonthermal effects. C. Temperature Rise Continuous exposure with a negligible temperature rise ( C) can be performed up to 4.5 W/kg for cell monolayers, but only up to 0.8 W/kg for cell suspensions (SAR average without meniscus). The small temperature rise of the monolayer exposure results from the low SAR of the medium (compared to the monolayer) and from the efficient cooling (maximum SAR is located at the medium/dish interface, where heat can be efficiently removed). It is both advantageous and unique that the monolayer exposure achieves the ICNIRP limit of 2 W/kg without introducing active liquid cooling based on an external medium at a different temperature. Active liquid cooling requires a considerable engineering effort to guarantee the same absolute temperature for the cells in sham and exposure and will additionally introduce temperature gradients in the medium. However, for experiments with SAR levels in the order of the occupational limits, i.e., at 10 W/kg or higher, a setup with active liquid cooling is required. Theoretically, the temperature gradient developed between the surface and bottom of the exposed cell medium for monolayer exposure can induce mass convection within the liquid volume. However, such a temperature gradient exists only for a short period of time because it is quickly equalized by heat conduction. Moreover, if it is combined with the low height of the medium, it is unlikely to result in mass movement. The product of the Grashof and Prandtl numbers is an indicator for liquid convection phenomena [4]. For a temperature gradient of 0.1 C over a 3-mm vertical distance inside the cell medium (3-mm medium height corresponds to the suggested sample volume of 3 mL for the Petri dishes), the value of the product is still approximately 11 times lower than the value required for the initiation of mass convection. D. Exposure and Environmental Control A high level of exposure and environmental control was realized. The feedback regulated exposure provides a low variability of SAR for the monolayer configuration (5.1%). Low variability is also present for the suspension exposure (5.9%), but only when water evaporation in the 60-mm Petri dish can be excluded. This can be achieved by using a high-humidity incubator environment or a cover for the 60-mm dish. If water evaporation is present, average variations of SAR are estimated to be in the order of 16% (Table VI). With respect to the environmental control, it is a great advantage of the presented setup that the exposure and sham units can be placed within the same commercial incubator providing the environmental conditions. The forced airflow exchange system allows excellent temperaC between two unexposed ture control with differences waveguides. VI. CONCLUSION An exposure system for conducting in vitro laboratory studies in several European research institutes was realized and characterized. The waveguide-based computer-controlled setup enables the exposure of cell monolayers and suspensions with an excellent SAR efficiency 50 and 10 (W/kg)/W, respectively. The flexible signal unit allows the generation and control of complex modulated signals, e.g., temporal changes between different GSM operation modes (DTX/non-DTX). The exposure field strength and environmental parameters (air temperature, fan system) are continuously monitored. The field information is used for feedback control. A coupled electrothermal FDTD analysis was performed and resulted in a nonuniformity of SAR of 30% and 40%. The temperature rise was assessed by measurement and simulation, and a maximum rise of 0.03 C/(W/kg) and 0.13 C/(W/kg) for monolayer and suspension was found. No localized temperature “hot spots” are generated within the cell medium. All simulations were verified by dosimetric measurements. 2066 IEEE TRANSACTIONS ON MICROWAVE THEORY AND TECHNIQUES, VOL. 52, NO. 8, AUGUST 2004 REFERENCES [1] A.-J. Chapman, Heat Transfer, 3rd ed. New York: Collier–Macmillian, 1974. [2] “ETSI-GSM recommendation,” ETSI, Sophia-Antipolis, France, Tech. Rep. GSM 05.05 prETS 300 577, 1996. [3] G.-B. Gajda, J.-P. McNamee, A. Thansandote, S. Boonpanyarak, E. Lemay, and P.-V. Bellier, “Cylindrical waveguide applicator for in vitro exposure of cell culture samples to 1.9 GHz radiofrequency fields,” Bioelectromagnetics, vol. 23, pp. 592–598, 2002. [4] J.-P. Holman, Heat Transfer, 8th ed. 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Erwin, “Dosimetry considerations in far field microwave exposure of mammalian cells,” Physiol. Chem. Phys. Med. Nuc. Magn. Resonance, vol. 20, pp. 23–30, 1998. [10] E.-G. Moros, W.-L. Straube, and W.-F. Pickard, “The radial transmission line as broad-band shielded exposure system for microwave irradiation of large numbers of culture flasks,” Bioelectromagnetics, vol. 20, pp. 65–80, 1999. [11] N. Nikoloski, J. Fröhlich, T. Samaras, J. Schuderer, and N. Kuster, “Dosimetric revaluation of the TEM cell setup for replication study,” Bioelectromagnetics, 2003, submitted for publication. [12] “Guidelines for evaluating and expressing the uncertainty of NIST measurement results,” NIST, Gaithersburg, MD, Tech. Rep. Tn1297, 1994. [13] M. Penafiel, T. Litovitz, D. Krause, A. Desta, and M. Mullins, “Role of modulation on the effect of microwaves on ornithine decarboxylase activity in L929 cells,” Bioelectromagnetics, vol. 18, pp. 132–141, 1997. [14] K. Poković, T. Schmid, and N. Kuster, “Millimeter-resolution E -field probe for isotropic measurement in lossy media between 100 MHz and 20 GHz,” IEEE Trans. Instrum. Meas., vol. 49, pp. 873–878, Aug. 2000. [15] T. Samaras, P. Regli, and N. Kuster, “Electromagnetic and heat computations for nonionizing radiation dosimetry,” Phys. Med. Biol., vol. 45, pp. 2233–2246, 2000. [16] F. Schönborn, K. Poković, M. Burkhardt, and N. Kuster, “Basis for optimization of in vitro exposure apparatus for health hazard evaluations of mobile communications,” Bioelectromagnetics, vol. 22, pp. 547–559, 2001. [17] F. Schönborn, K. Poković, A. Wobus, and N. Kuster, “Design, optimization, realization, and analysis of an in vitro system for the exposure of embryonic stem cells at 1.71 GHz,” Bioelectromagnetics, vol. 21, pp. 372–384, 2000. [18] J. Schuderer and N. Kuster, “The effect of the meniscus at the solid/liquid interface on the SAR distribution in petri dishes and flasks,” Bioelectromagnetics, vol. 24, pp. 103–108, 2003. [19] J. Schuderer, D. Spät, T. Samaras, W. Oesch, and N. Kuster, “In vitro exposure systems for RF exposures at 900 MHz,” IEEE Trans. Microwave Theory Tech., vol. 52, pp. 2067–2075, Aug. 2004. Jürgen Schuderer was born in 1972. He received the M.S. degree in physics from the University of Freiburg, Freiburg, Germany, in 1999, and the Ph.D. degree from the Swiss Federal Institute of Technology (ETH), Zürich, Switzerland, in 2003. He was with the Fraunhofer Institute for Physical Measurement Techniques, Freiburg, Germany, where he developed a fluoroptic sensor system to monitor bioaffinity reactions. In late 1999, he joined the Integrated Systems Laboratory, ETH, where he was involved with the development of in vitro and humanexposure setups for risk-assessment studies, as well as with the development of miniaturized dosimetric sensors. In 2003, he joined the Foundation for Research on Information Technologies in Society (IT’IS), Zürich, Switzerland. His research interest is currently focused on RF dosimetry, biomedical sensors, and computational electrodynamics. Theodoros Samaras (S’93–A’97–M’02) was born in 1968. He received the Physics degree from the Aristotle University of Thessaloniki, Thessaloniki, Greece, in 1990, the M.Sc. degree in medical physics (with distinction) from the University of Surrey, Surrey, U.K., in 1991, and the Ph.D. degree in hyperthermia from the Aristotle University of Thessaloniki, in 1996. Following his military service, he joined the Federal Institute of Technology (ETH), Zürich, Switzerland, where he was involved with the modeling of the interaction of electromagnetic waves with biological tissues. Since December 1999 he has been working in the Department of Physics of the Aristotle University of Thessaloniki as a Lecturer. His research interests include computational electromagnetics, microwave applications, and biomedical engineering. Dr. Samaras is a member of the European Society for Hyperthermic Oncology (ESHO). In February 1999, he was the recipient of a Marie-Curie Fellowship presented by the European Commission to work on the enhancement of superficial hyperthermia in the Hyperthermia Section, University Hospital Rotterdam-Daniel. Walter Oesch was born in 1974. He received the M.S. degree in geophysics from the Swiss Federal Institute of Technology (ETH), Zürich, Switzerland, in 2000 . He then joined the Foundation for Research on Information Technologies in Society (IT’IS), Zürich, Switzerland. He specified, planned, designed, and implemented the controlling and monitoring software for various in vivo and in vitro exposure setups. His primary research interest is concentrated in the area of technical software engineering. Denis Spät was born in 1976. He received the M.S. degree in industrial engineering from the Technical University of Darmstadt, Darmstadt, Germany, in 2003. Since 2002, he has been a Scientific Assistant with the Foundation for Research on Information Technologies in Society (IT’IS), Zürich, Switzerland. His main research interest is RF dosimetry for bioexperiments focusing on the health-risk assessment of electromagnetic-field exposures. Niels Kuster (M’93) was born in Olten, Switzerland, in 1957. He received the M.S. and Ph.D. degrees in electrical engineering from the Swiss Federal Institute of Technology (ETH), Zürich, Switzerland. In 1993, he became a Professor with the Department of Electrical Engineering, ETH. In 1992, he was an Invited Professor with the Electromagnetics Laboratory, Motorola Inc., Fort Lauderdale, FL, and in 1998, with the Metropolitan University of Tokyo, Tokyo, Japan. In 1999 he became Director of the Foundation for Research on Information Technologies in Society (IT’IS), Zürich, Switzerland. His research interest is currently focused on the area of reliable on/in-body wireless communications and related topics. This includes measurement technology and computational electrodynamics for evaluation of close near-fields in complex environments, safe and reliable wireless communication links within the body or between implanted devices and the outside for biometrics applications, development of exposure setups and quality control for bioexperiments evaluating interaction mechanisms, therapeutic effects, as well as potential health risks, and exposure assessments. Dr. Kuster is a member of several standardization bodies and has consulted several government agencies on the issue of the safety of mobile communications. He also served on the boards of scientific societies, research management councils for governments, and editorial boards.