**4. The concept of real-time acquisitions in bioelectromagnetic investigations**

Real-time data acquisition in biomedicine is employed in a lot of different applications, both *in vitro* and *in vivo*. Real-time monitoring of chemical reactions (Shutes and Der, 2005), gene expression (Gubern et al., 2009), drug release from nanoparticles (Chouhan and Bajpai, 2009) are significant examples of *in vitro* applications. As for *in vivo* investigations, the real-time acquisition is usually based on non-invasive imaging techniques (Ohtani et al., 2010; Li et al., 2010; Voyvodic et al., 2011) suitable to be applied to living animals.

The common issue in both *in vitro* and *in vivo* real-time experiments for biomedicine is that the experimental acquisition of physiological data is performed simultaneously with the correlated event.

Real Time Radio Frequency Exposure for Bio-Physical Data Acquisition 299

Fig. 2. Single channel current recorded before, during, and after the exposure to RF field

**SAMPLE OBSERVABLE EXPOSURE TECHNIQUE/** 

Heart slices Field potential Pulsed 9.2 GHz Extracellular

Skeletal muscle Muscle contraction CW 0.75-1.12 GHz Force transducer

Liposomes Enzymatic activity CW 2.45 GHz Spectrophotometer Table 1. Schematic description of the *in vitro* real-time experiments in the literature, in terms of samples, observables, RF signals and techniques used for the experimental activity

The most used experimental samples are excitable cells and tissues since they are likely to be sensitive to EM stimulation and to display reversible effects. They include single neuronal cells (Liberti et al., 2004; Platano et al., 2006; Marchionni et el., 2005; Paffi et al., 2007), neuronal networks (Tattersall et al., 2001; Pakhomov et al., 2003; Koester et al., 2007; Merla et al., 2011), inner ear hair cells of the organ of Corti (El Ouardi et al., 2011), and muscles, both skeletal (Lambrecht et al., 2006) and cardiac (Pakhomov et al., 2000). The observables are mostly electrophysiological parameters, such as the ionic current and the membrane voltage at the level of single cell, or the field potential generated by a group of interconnected neuronal cells. This latter observable is largely used in the studies on synaptic plasticity, the basic mechanism underlying high cognitive functions, such as memory and learning. However, even chemical parameters can be analyzed, such as the enzymatic activity, through a spectrophotometer (Ramundo-Orlando et al., 2004), or the neurotransmitter release, through an electrochemical detector (Hagan et al., 2004; Yoon et al., 2006). The techniques employed for the acquisition of the electrophysiological traces are patch-clamp (Neher and Sakmann, 1998) and extracellular recordings. Both of them require the use of microelectrodes near the membrane cell (extracellular recordings) or in close contact with it (patch-clamp). The EM signals mostly used in the experiments are

CW 900; 1800 MHz;

GSM900

UMTS

CW 700 MHz; Pulsed 9.2 GHz; CW 1.8 GHz

CW 1-6 GHz

**INSTRUMENT** 

Patch-clamp recording

Patch-clamp recording

Extracellular recording

recording

Electrochemical detector

published *in vitro* real-time experiments is summarized.

Membrane voltage

Organ of Corti Ionic current GSM900; GSM1800;

Chromaffin cells Catecholamine release CW 0.75-1.12 GHz;

Neuronal cells Ionic current;

Brain slices Field potential

Real-time experiments have revealed a powerful tool especially in the investigation on possible modifications of the activity of neuronal cells or cell networks during the exposure, through electrophysiological recordings, as evident from Table 1, where information on

In bioelectromagnetic investigation the concept of real-time is applied to experimental acquisition of data, which have to be collected simultaneously with the EM exposure, as schematically reported in Fig. 1.

In real-time experiments, the time slot of data acquisition often starts before and ends after the exposure period. The data acquired before and after the exposure are compared with those collected during the exposure. The pre-exposure data are used as a negative control; the post-exposure ones are used to identify possible long term or permanent modifications induced by the RF field on the observed parameter. Conversely, in off-line experiments data acquisition is carried out at the end of the exposure (Fig. 1) and the negative control is obtained from a parallel session (sham exposure) where biological samples are subjected to environmental conditions identical to those of the group of the exposed ones, except for the exposure. On the contrary, in real-time experiments, the same sample, observed in different time slots, is used as its own control. This concept is better explained in Fig. 2, showing the current through an ionic channel recorded before (black trace on the left), during (red trace on the center) and after (black trace on the right) the exposure.

From Fig. 2, a clear effect of the RF field is observable only comparing the three segments of the recorded current. Being reversible, such an effect would not be detected in off-line experiments.

Therefore, the main advantage of real-time acquisition is that it enables the experimenter to identify possible reversible or cumulative effects, otherwise difficult or even impossible to be detected. Moreover real-time experiments permit time saving, since one does not have to wait for the end of the experiment before collecting and analyzing the biological data.

Fig. 1. Schematic representation of the time scheduling of exposure and data acquisition in real-time and off-line experiments

298 Real-Time Systems, Architecture, Scheduling, and Application

In bioelectromagnetic investigation the concept of real-time is applied to experimental acquisition of data, which have to be collected simultaneously with the EM exposure, as

In real-time experiments, the time slot of data acquisition often starts before and ends after the exposure period. The data acquired before and after the exposure are compared with those collected during the exposure. The pre-exposure data are used as a negative control; the post-exposure ones are used to identify possible long term or permanent modifications induced by the RF field on the observed parameter. Conversely, in off-line experiments data acquisition is carried out at the end of the exposure (Fig. 1) and the negative control is obtained from a parallel session (sham exposure) where biological samples are subjected to environmental conditions identical to those of the group of the exposed ones, except for the exposure. On the contrary, in real-time experiments, the same sample, observed in different time slots, is used as its own control. This concept is better explained in Fig. 2, showing the current through an ionic channel recorded before (black trace on the left), during (red trace

From Fig. 2, a clear effect of the RF field is observable only comparing the three segments of the recorded current. Being reversible, such an effect would not be detected in off-line

Therefore, the main advantage of real-time acquisition is that it enables the experimenter to identify possible reversible or cumulative effects, otherwise difficult or even impossible to be detected. Moreover real-time experiments permit time saving, since one does not have to wait for the end of the experiment before collecting and analyzing the biological data.

Fig. 1. Schematic representation of the time scheduling of exposure and data acquisition in

on the center) and after (black trace on the right) the exposure.

schematically reported in Fig. 1.

real-time and off-line experiments

experiments.

Fig. 2. Single channel current recorded before, during, and after the exposure to RF field

Real-time experiments have revealed a powerful tool especially in the investigation on possible modifications of the activity of neuronal cells or cell networks during the exposure, through electrophysiological recordings, as evident from Table 1, where information on published *in vitro* real-time experiments is summarized.


Table 1. Schematic description of the *in vitro* real-time experiments in the literature, in terms of samples, observables, RF signals and techniques used for the experimental activity

The most used experimental samples are excitable cells and tissues since they are likely to be sensitive to EM stimulation and to display reversible effects. They include single neuronal cells (Liberti et al., 2004; Platano et al., 2006; Marchionni et el., 2005; Paffi et al., 2007), neuronal networks (Tattersall et al., 2001; Pakhomov et al., 2003; Koester et al., 2007; Merla et al., 2011), inner ear hair cells of the organ of Corti (El Ouardi et al., 2011), and muscles, both skeletal (Lambrecht et al., 2006) and cardiac (Pakhomov et al., 2000). The observables are mostly electrophysiological parameters, such as the ionic current and the membrane voltage at the level of single cell, or the field potential generated by a group of interconnected neuronal cells. This latter observable is largely used in the studies on synaptic plasticity, the basic mechanism underlying high cognitive functions, such as memory and learning. However, even chemical parameters can be analyzed, such as the enzymatic activity, through a spectrophotometer (Ramundo-Orlando et al., 2004), or the neurotransmitter release, through an electrochemical detector (Hagan et al., 2004; Yoon et al., 2006). The techniques employed for the acquisition of the electrophysiological traces are patch-clamp (Neher and Sakmann, 1998) and extracellular recordings. Both of them require the use of microelectrodes near the membrane cell (extracellular recordings) or in close contact with it (patch-clamp). The EM signals mostly used in the experiments are

Real Time Radio Frequency Exposure for Bio-Physical Data Acquisition 301

Before carrying out the experimental activity, a hypothesis is formulated (step 1), leading to the choice of an appropriate biological system to be exposed. It can be a particular kind of cell, a tissue, an organ, or a whole organism, as an animal. The experiment to test the hypothesis is then defined (step 2), including biological models, endpoints, techniques, and exposure parameters. The outcome of the analyses carried out during this step determines all the requirements, both biological and EM, of the exposure system. Moving from biological protocol and requirements on exposure parameters, the most suitable EM basic structure is chosen (step 3). If a system meeting all the requirements for the planned experiment is already present in the literature, one may decide to use that system as it is or to adapt it to new constrains; otherwise, the chosen EM structure must be first dimensioned on theoretical bases (step 4). Generally, the basic structure must be modified in order to meet all the biological and EM requirements. Thus, an iterative process of adaptation and optimization of the system (step 5) takes place using numerical tools, leading to the nal design parameters (dimensions, materials, sample position, etc.). The next two steps are the manufacture (step 6) and the experimental validation (step 7) of the exposure system. Measurements should be conducted firstly with the empty structure; then it has to be loaded with the biological sample to validate the behavior of the system and to experimentally evaluate the dosimetry, i.e. the SAR distribution in the sample. If acceptable agreement between measurement and simulation is not achieved, one must return to steps 5, 6, or 7

As already noted, biological requirements represent a crucial point for the design of an exposure system since they could be the most limiting ones, especially when a particular

Although the procedure (Paffi et al., 2010) for the design and characterization of the exposure system is the same regardless of the particular kind of analysis (real-time or offline), real-time acquisitions impose additional biological requirements to the system. In particular, when dealing with the electrophysiological recordings, they can be summarized in the easy access to the sample (i.e. through the microscope, the electrodes, and the perfusion apparatus) and the minimum coupling between the EM field and the acquisition

For the correct placement of the electrodes, the sample must be illuminated and observed through a microscope. Moreover, in some experiments, the biological sample, e.g. brain slices, has to be perfused to preserve its structure and function. For these reasons, in order to easily reach the sample, the exposure system must be either an open structure or a closed

The microelectrodes for electrophysiological recordings are usually made of glass filled with a saline solution with a metallic wire inside. Being the biological cultures exposed to CW or modulated signals, especially at the frequencies typical of mobile communication, the realtime acquisition implies the possibility of EM coupling between the electrodes and the electric field, with consequent artifacts on the recorded trace. Another possible source of artifacts is the interference between the EM field generated by the system and the whole acquisition setup. To minimize the coupling between the field and the electrode, the direction of the electric eld

equipment and protocol procedures are needed, e.g. in real-time experiments.

**5.1 Special requirements for the design of real-time systems** 

depending on the hypothesized cause of mismatch.

equipment, in order to avoid artifacts.

one modified with holes.

continuous waves (CW) or modulated signals, especially at the frequencies typical of mobile communication. In one case (Pakhomov et al., 2003), the effects of a pulsed signal at 9 GHz on brain slices were investigated.

Due to the complexity of the experimental procedure, generally exposure systems for realtime experiments are more difficult to design since, beside the requirements typical of any exposure system (see Section 2), they have to meet additional and demanding requirements (see Section 5).

In particular, for *in vivo* experiments, only two real-time systems are present in the literature from 1999 until now (Testilier et al., 2002; Arima et al., 2011). Both of them are based on a simple EM structure (i.e. an antenna), while the real-time monitoring of the biological parameter is obtained through highly invasive techniques. In Testilier et al. (2002) neurotransmitter levels in a rat brain were monitored during the exposure through an invasive microdialysis technique; in Arima et al. (2011) the microcirculation of the rat brain during the exposure is observed through a cranial window on the skull.

The reason of such a reduced number of *in vivo* studies can be found in bioassays typically used to measure the effects of EM fields that require the employment of complex procedures and instrumentation. Biological data are often collected after the animal sacrifice and the subsequent removal of organs and tissues for the analysis.
