**6.1 General classification**

Due to the great variety of biological targets and protocols, a number of different exposure systems have been adopted in the experimental investigation, both *in vivo* and *in vitro*.

Despite their peculiarities, the exposure systems can be classified on the basis of the employed EM structure into three main groups: radiating, propagating and resonant systems, as proposed in (Lovisolo et al., 2009; Paffi et al., 2010).

Conversely, for what concerns the biological experiment, the main classification is between the *in vitro* and *in vivo* setups, as already discussed in Section 3.

In turn, with respect to the experimental protocol, *in vivo* systems have been classified in two main classes, depending on whether the exposure involves the whole body or is focalized into a specific organ (Paffi et al., 2011). An additional criterion of organization is the possibility for the animals to freely move during the exposure (Paffi et al., 2011). In this case, animals are generally housed in their own cages, provided with food and water. 302 Real-Time Systems, Architecture, Scheduling, and Application

must be almost orthogonal to the electrode. Moreover, to avoid interference of the eld with the laboratory equipment, in principle, the EM field should be confined in a closed structure. However, even open structures can be used, provided that the electric and magnetic fields

Finally, the presence of metallic and/or dielectric objects (microscope objective, lamp, electrodes, perfusion apparatus), placed very close to the region where the sample is exposed (see Figure 4), may modify the field distribution and thus the whole behavior of the system. This kind of coupling must be carefully taken into account during the numerical

The different solutions adopted to meet such demanding requirements will be described in

Fig. 4. Picture of a planar exposure system used for electrophysiological recordings from brain slices (Paffi et al., 2007). Arrows highlight the presence of different objects placed very

Due to the great variety of biological targets and protocols, a number of different exposure systems have been adopted in the experimental investigation, both *in vivo* and *in vitro*.

Despite their peculiarities, the exposure systems can be classified on the basis of the employed EM structure into three main groups: radiating, propagating and resonant

Conversely, for what concerns the biological experiment, the main classification is between

In turn, with respect to the experimental protocol, *in vivo* systems have been classified in two main classes, depending on whether the exposure involves the whole body or is focalized into a specific organ (Paffi et al., 2011). An additional criterion of organization is the possibility for the animals to freely move during the exposure (Paffi et al., 2011). In this case, animals are generally housed in their own cages, provided with food and water.

sharply decay with the distance from the system.

optimization of the system in order to minimize it.

close to the system surface during the experiments.

**6. Classification of the exposure systems** 

systems, as proposed in (Lovisolo et al., 2009; Paffi et al., 2010).

the *in vitro* and *in vivo* setups, as already discussed in Section 3.

**6.1 General classification** 

Section 6.

Otherwise animals are restrained inside plastic holders, to guarantee their relative position and orientation with respect to the electric and magnetic fields.

Conversely, *in vitro* exposure systems have been classified (Paffi et al., 2010) in two different groups: off-line and real-time, depending on the kind of data acquisition they have been designed for. This latter classification has not been adopted for the *in vivo* setups, since realtime acquisitions are very uncommon for *in vivo* experiments, as pointed out in Section 4.

Proposed classification is reported in Figure 5, together with the number of systems belonging to each category and published in international journals since 1999.

Fig. 5. Classification of the exposure systems. For each category the number of systems published from 1999 is reported

Among the 52 *in vivo* systems, those used for local exposure (19) are exclusively based on radiating structures. In particular, they are small antennas placed close to the target organ (brain, ear, eye) to induce significant and localized power absorption (Paffi et al., 2011). To reduce the cost of the experiment, a single antenna can be used to simultaneously expose several animals, if they are arranged in a sort of carousel around it (Schönborn et al., 2004). Generally, animals locally exposed are restrained within plastic holders to obtain a more accurate and precise SAR distribution, even though body-mounted antennas (Bahr et al., 2007) become necessary for the well being of animals when the exposure is prolonged.

For the 33 whole body exposure setups, the uniformity of SAR absorbed by animals of the same group and within each animal was a critical requirement (Paffi et al., 2011). This is particularly difficult to achieve especially for large-scale experiments. In this case, radiating structures (14 systems found in the literature) could be particularly suitable since a lot of bodies can be simultaneously exposed to a plane-wave equivalent field (Paffi et al., 2011).

Real Time Radio Frequency Exposure for Bio-Physical Data Acquisition 305

According to the classification described above, real-time *in vitro* systems published in international journals from 1999 up to now, have been organized in a schematic view in

*Modified rectangular* 

*RADIATING Horn antenna* (Yoon et al., 2006).

Propagating systems for real-time studies are generally closed structures modied with holes for sample observation and perfusion, and for online monitoring of biochemical or

They are mostly TEM cells and rectangular waveguides as described in (Linz et al., 1999), where the electrophysiological activity of myocyte cells was recorded, through the patchclamp technique, during the exposure to 900 MHz and 1800 MHz CW. The systems were equipped with two holes: one in the top wall to insert the recording electrode and the other in the bottom to observe the sample with a microscope. The solution adopted to avoid interference between the electric eld inside the guide and the metallic wire of the patchclamp electrode was the elongation of the glass microelectrodes used for sealing the cell in order to place the wire outside the exposure device. The power efciency was not bad: 1.66

A rectangular waveguide (Lambrecht et al., 2006), operating between 0.75–1.12 GHz, was also used to measure, through a force transducer, the effects of the RF field on skeletal muscle inserted in a bath placed in the center of the waveguide. The waveguide was modified with slots in the walls to allow connection with measurement, control, and stimulating devices outside the system. The power efciency was good (> 3 (W/kg)/W) and SAR homogeneity, around 79 %, was in line with minimal requirements for *in vitro* experiments (homogeneity > 70 %). A modied rectangular waveguide was used for delivering CW or Universal Mobile Telecommunications System (UMTS) signals to neuronal networks during electrophysiological recordings from multiple electrodes (Koester et al.,

*waveguide* 

Table 2. Classification of the real-time *in vitro* systems published from 1999

*RESONANT Modified rectangular* 

(W/kg)/W at 900 MHz and 3.16 (W/kg)/W at 1800 MHz.

**EXPOSURE SYSTEM REFERENCE** 

*Modified TEM cell* (Linz et al., 1999);

*Parallel plates* (Tattersall et al., 2001). *Coplanar waveguide* (Liberti et al., 2004);

*Fin-line* (El Ouardi et al., 2011).

*waveguide* (Hagan et al., 2004).

(Merla et al., 2011).

(Linz et al., 1999); (Pakhomov et al., 2000); (Lambrecht et al., 2006); (Koester et al., 2007).

(Paffi et al., 2007).

*Modified stripline* (Ramundo-Orlando et al., 2004).

**6.2 Real time systems in the literature** 

*PROPAGATING* 

**6.2.1 Propagating structures** 

biophysical parameters.

Table 2.

**REAL-TIME** 

However, the power efficiency is quite low (Paffi et al., 2011). Propagating systems are mainly based on rectangular or radial waveguides (Hansen et al., 1999), whereas resonant cavities are mainly obtained by shorting radial waveguides through metallic rods (Balzano et al., 2000). These structures are characterized by high volume efficiency, i.e. a lot of bodies (up to 65 animals) can be simultaneously exposed in a limited space. However, due to the importance of a correct body positioning, animals are restrained within plastic cylinders. Therefore, resonant cavities are suitable for chronic long-term (days or moths) exposures only if the exposure is limited to a few hours per day, as done in some two years bioassay experiments (e.g. PERFORM A).

A separated mention must be made of reverberating chambers (Corona et al., 2001). Inside those systems a statistically uniform field can be obtained and a large number of animals can be simultaneously exposed in a habitat simulating the usual one of animals (Wu et al., 2010). These are the reasons why they are suitable for large-scale, long-term experiments.

Moving to the *in vitro* systems, among the 54 off-line setups found in the literature, the most used (24) are propagating structures (Paffi et al., 2010), such as Transverse Electromagnetic (TEM) cells and rectangular waveguides. The main advantages of propagating structures are the EM field uniformity inside the biological sample and versatility. With a proper dosimetric characterization, propagating systems have been used to expose different volumes of various kinds of cells inside sample holders, such as multiwells, Petri dishes, and flasks (Paffi et al., 2010). Also resonant systems have been largely used (12) in off-line *in vitro* experiments (Paffi et al., 2010). They are closed and compact structures, such as shortcircuited waveguides; thus both the active and the sham systems easily fit inside a commercial incubator, often needed for maintaining the optimal environmental conditions (Schuderer et al., 2004). Due to the onset of a standing wave inside resonant systems, the power efficiency is generally high, but the positioning of the sample is critical, because of the extremely localized regions of field uniformity (Paffi et al., 2010). On the contrary, radiating systems, usually consisting of commercial or ad hoc fabricated antennas, present low power efficiency, but generally allow for the simultaneous exposure of a lot of samples. However, they need EM compatible arrangements due to the lack of enclosures confining the emitted field (Paffi et al., 2010).

Regarding the 13 real-time *in vitro* systems, they are mostly based on propagating structures, with the only exception of one resonant (Hagan et al., 2004) and one radiating system (Yoon et al., 2006). Indeed, propagating structures are generally the most versatile and adaptable to the additional constrains imposed by real-time acquisition.

To meet such requirements, two main solutions have been proposed in the literature (Paffi et al., 2010): closed structures modified with holes for sample observation and data recording and open systems specially designed to have the field confined in a small volume around the surface. This latter solution also implies high values of power efficiency (Liberti et al., 2004; Paffi et al., 2007).

A detailed description of different kinds of real-time systems, together with their main features, will be given in Section 6.2.
