**3.1. Impact on living cells**

274 Ultra-Wideband Radio Technologies for Communications, Localization and Sensor Applications

**Figure 15.** Measured reflection coefficient of the MR-compatible double-ridged horn antenna (lower

Fig. 15 displays measured results for the reflection coefficient and the gain of the modified DRH antenna. A return loss above 10 dB was achieved over the frequency range from 1.5 to 12 GHz. Radiation measurements in an anechoic chamber yielded the radiation patterns illustrated in Fig. 16 for two orthogonal cuts with respect to the plane of the ridges. The halfpower beam width, indicated as the black contour line in Fig. 16, was found to vary between 30 and 50 degrees, thus covering a range suitable for the envisaged applications. Except for frequencies around 2 GHz, the main lobe showed little spectral variation. The corresponding frequency variation of the antenna gain is displayed in Fig. 16. These results

**Figure 16.** Two-dimensional representation of the measured radiation pattern of the MR-compatible double-ridged horn antenna for the E-plane (left) and the H-plane (right) through the main beam. The scales indicate the antenna gain in dBi. The black and white contour lines illustrate the corresponding

The transient response of the antenna is shown in Fig. 17. Despite the open geometry of the MR-compatible antenna, a low signal distortion could be sustained. The slight angular dependence of the time responses can be attributed to an offset between the phase centers of

beam widths at 3 and 10 dB below the frequency-dependent maximum gain, respectively.

the antennas and the center of rotation of the antenna positioning system.

curve) and the measured antenna gain (upper curve) versus frequency.

were found in good agreement with the numerical simulations.

The electrical properties of biological tissues and cell suspensions have been of interest for over a century for many reasons. They determine the pathways of current flow through the body and are very important for the analysis of a wide range of biomedical applications such as functional electrical stimulation and the diagnosis and treatment of various physiological conditions with weak electric currents, radio-frequency hyperthermia, electrocardiography, and body composition. On a more fundamental level, the knowledge of these electrical properties can lead to an understanding of the underlying basic biological processes. Indeed, biological impedance studies have long been an important issue in electrophysiology and biophysics; interestingly, one of the first demonstrations of the existence of the cell membrane was based on dielectric studies on cell suspensions [33].

Biological tissues are a mixture of water, ions, and organic molecules organized in cells, subcellular structures, and membranes, and its dielectric properties are highly frequencydependent in the range from Hz to GHz. The spectrum is characterized by three main dispersion regions referred to as α, β, and γ regions at low, intermediate, and high frequencies [34]. Biological materials can show large dispersions, especially at low frequencies (Fig. 18). Low frequencies are mainly caused by interfacial polarizations at the surfaces between the different materials of which a cell is composed [35]. Reviews of the dielectric properties of cells and the different dispersions are given in the literature [36], [37].

**Figure 18.** Spectrum of the dielectric properties of cell suspensions and tissues.

The step changes in *<sup>r</sup>* are called dispersions and are due to the loss of particular polarization processes as frequency increases. The α-dispersion is due to the flow of ions across cell surfaces, the β-dispersion results from the charge at cell membranes, the δdispersion is produced by the rotation of macromolecular side-chains and bound water, and the γ-dispersion is due to the dipolar rotation of small molecules particularly water [35] (figure reproduced with permission from Elsevier).

When exposed to electric fields, living cells behave as tiny capacitors, accumulating charges on the cell surface. The permittivity of living cell suspensions is dependent on the frequency, and falls in a series of the dispersions described above, as frequency increases. The β-dispersion, between 0.1 and 100 MHz, results from the build-up of charges at cell membranes. The difference between permittivity measurements made at two frequencies, on either side of the β-dispersion range, is proportional to the viable biomass concentration. With spherical cells, the permittivity increment is given by equation [38].

$$
\Delta \varepsilon = \frac{\text{9 } P \text{ } r \text{ } \mathbb{C}\_m}{4} \tag{1}
$$

ultraMEDIS – Ultra-Wideband Sensing in Medicine 277

plates were placed in a Faraday cage (to avoid any irradiation). After continued incubation for 24, 48 and 72 h, the vitality of cells was determined by colorimetric identification (MTT assay for measuring the activity of enzymes that reduce MTT [3-(4.5-Dimethylthiazol-2-yl)- 2.5-diphenyltetrazolium bromide, yellow tetrazole] to formazan, giving a purple color). The measured vitality of control cells was normalized to 100%, and the vitality of exposed cells was put into relation. The vitality of exposed cells was related to non-exposed cells. Due to biological fluctuations, data between 70% and 120% vitality were assessed as not influenced. As depicted in Fig. 19, none of the determined cells was influenced by ultra-wideband

**Figure 19.** Impact of ultra-wideband electromagnetic waves on the vitality of living cells. The upper part of the figure shows light images of the fibroblast cell line BJ and the cancerous cell line BT474. The lower figure depicts the vitality of the fibroblasts BJ and the cancerous cells BT474 after UWB exposition with 4 mW for 5, 30 or 60 min. The vitality was observed 0, 24, 48 or 72 h after exposure. The depicted vitality of exposed cells is related to non-exposed cells. Due to biological fluctuations, data between 80%

The electrical properties of tissues and cell suspensions are most unusual. They change with frequency in three distinct steps (dispersions as described above) and their dielectric constants reach enormous values at low frequencies. Extensive measurements were carried out over a broad frequency range extending from less than 1 Hz to many GHz. The observed frequency changes of these properties obey causality, i.e., the Kramers-Kronig relationships which relate changes of dielectric constants with conductivity changes. A number of mechanisms were identified which explain the observed data. These mechanisms reflect the various compartments of the biological material. These include membranes and

and 120% vitality was not considered to be cytotoxic [25].

**3.2. Animal tissue** 

electromagnetic waves.

As long as there is no change in the cell radius *r* or the membrane capacitance *Cm* , the permittivity increment is proportional to the cell volume fraction *P* [39].

As a starting point for developing new applications, it is critical to characterize differences in the dielectric properties of the cells, for example human leukocyte subpopulations [40]. Even though, a comparative analysis of the dielectric properties of the cells is necessary, and the UWB radiation on cells itself has to be characterized, too. For this reason, experiments with two different cell lines (tumor cell line BT474 and fibroblasts BJ) were performed. Cell suspensions of these cell lines were disseminated, and the growth rate was determined. Afterwards, the cells were seeded on 96-well plates, cultivated for 24 h and exposed to UWB radiation *via* UWB-M-sequence radar with double-ridged horn antennas of about 10 dBi average gain for 5, 30 or 60 min. As non-treated control, for the same time, plates were placed in a Faraday cage (to avoid any irradiation). After continued incubation for 24, 48 and 72 h, the vitality of cells was determined by colorimetric identification (MTT assay for measuring the activity of enzymes that reduce MTT [3-(4.5-Dimethylthiazol-2-yl)- 2.5-diphenyltetrazolium bromide, yellow tetrazole] to formazan, giving a purple color). The measured vitality of control cells was normalized to 100%, and the vitality of exposed cells was put into relation. The vitality of exposed cells was related to non-exposed cells. Due to biological fluctuations, data between 70% and 120% vitality were assessed as not influenced. As depicted in Fig. 19, none of the determined cells was influenced by ultra-wideband electromagnetic waves.

**Figure 19.** Impact of ultra-wideband electromagnetic waves on the vitality of living cells. The upper part of the figure shows light images of the fibroblast cell line BJ and the cancerous cell line BT474. The lower figure depicts the vitality of the fibroblasts BJ and the cancerous cells BT474 after UWB exposition with 4 mW for 5, 30 or 60 min. The vitality was observed 0, 24, 48 or 72 h after exposure. The depicted vitality of exposed cells is related to non-exposed cells. Due to biological fluctuations, data between 80% and 120% vitality was not considered to be cytotoxic [25].

### **3.2. Animal tissue**

276 Ultra-Wideband Radio Technologies for Communications, Localization and Sensor Applications

**Figure 18.** Spectrum of the dielectric properties of cell suspensions and tissues.

With spherical cells, the permittivity increment is given by equation [38].

polarization processes as frequency increases. The α-dispersion is due to the flow of ions across cell surfaces, the β-dispersion results from the charge at cell membranes, the δdispersion is produced by the rotation of macromolecular side-chains and bound water, and the γ-dispersion is due to the dipolar rotation of small molecules particularly water [35]

When exposed to electric fields, living cells behave as tiny capacitors, accumulating charges on the cell surface. The permittivity of living cell suspensions is dependent on the frequency, and falls in a series of the dispersions described above, as frequency increases. The β-dispersion, between 0.1 and 100 MHz, results from the build-up of charges at cell membranes. The difference between permittivity measurements made at two frequencies, on either side of the β-dispersion range, is proportional to the viable biomass concentration.

9

As long as there is no change in the cell radius *r* or the membrane capacitance *Cm* , the

As a starting point for developing new applications, it is critical to characterize differences in the dielectric properties of the cells, for example human leukocyte subpopulations [40]. Even though, a comparative analysis of the dielectric properties of the cells is necessary, and the UWB radiation on cells itself has to be characterized, too. For this reason, experiments with two different cell lines (tumor cell line BT474 and fibroblasts BJ) were performed. Cell suspensions of these cell lines were disseminated, and the growth rate was determined. Afterwards, the cells were seeded on 96-well plates, cultivated for 24 h and exposed to UWB radiation *via* UWB-M-sequence radar with double-ridged horn antennas of about 10 dBi average gain for 5, 30 or 60 min. As non-treated control, for the same time,

4 *<sup>m</sup> PrC* 

is proportional to the cell volume fraction *P* [39].

are called dispersions and are due to the loss of particular

(1)

The step changes in *<sup>r</sup>*

permittivity increment

(figure reproduced with permission from Elsevier).

The electrical properties of tissues and cell suspensions are most unusual. They change with frequency in three distinct steps (dispersions as described above) and their dielectric constants reach enormous values at low frequencies. Extensive measurements were carried out over a broad frequency range extending from less than 1 Hz to many GHz. The observed frequency changes of these properties obey causality, i.e., the Kramers-Kronig relationships which relate changes of dielectric constants with conductivity changes. A number of mechanisms were identified which explain the observed data. These mechanisms reflect the various compartments of the biological material. These include membranes and

### 278 Ultra-Wideband Radio Technologies for Communications, Localization and Sensor Applications

their properties, biological macromolecules and fluid compartments inside and outside membranes [41]. Special topics include a summary of the significant advances in theories on counter ion polarization effects, dielectric properties of cancer *vs.* normal tissues, properties of low-water-content tissues [42], and macroscopic field-coupling considerations. The dielectric properties of tissues are often summarized as empirical correlations with tissue water contents in other compositional variables. The bulk electrical properties of tissues are needed for many bioengineering applications of electric fields or currents, and they provide insight into the basic mechanisms that govern the interaction of electric fields with tissue [43].

ultraMEDIS – Ultra-Wideband Sensing in Medicine 279

part of permittivity was determined in a

Gram-positive) of the Gram-positive

Gram-negative). From each strain

of both Gram-positive strains

respective research and application field, bacterial strains are currently detected by complex methods, for example: polymerase chain reaction (technique to amplify a single or a few copies of a piece of DNA), fluorescent *in situ* hybridization, DNA microarray and Raman-

Different studies have shed some light into the biomass determination of different microbial suspensions *via* dielectric spectroscopy. Mishima *et al.* investigated growth kinetics of bacterial, yeast and animal cells by dielectric monitoring in the frequency range of 10 kHz - 10 MHz [50]. The determination of bacterial growth by dielectric measurements was also shown by Harris *et al.* [51]. Jonsson *et al.* measured the concentration of bacterial cells *via* indirect methods based on the dielectric determination of ions in the suspension, which are released by killed cells [52]. Benoit *et al.* showed that it is possible to discriminate the hydrophobic or hydrophilic features of bacterial suspensions by determining the dielectric permittivity [53]. Nevertheless, no data are available for discrimination on the basis of bacterial structures *per se*, such as the presence of Gram-positive or Gram-negative bacterial

Therefore, two different Gram-positive bacterial strains (*Micrococcus luteus* and *Bacillus subtilis*) and two Gram-negative bacterial strains (*Escherichia coli* and *Serratia marcescens*) were cultivated under standard conditions using Standard I media and shaking flasks. Bacterial strains were incubated for 24 h at 37°C in an incubation shaker. To assess whether the Gram-status of bacteria could be determined by dielectric spectroscopy, bacterial suspensions were transferred to 50 ml tubes and centrifuged. The supernatant (liquid above precipitate) was removed, the pellet was washed in 0.9% sodium chloride solution and, finally, the dielectric properties of the bacterial biomass (pellet of 10 ml) were determined. Dielectric spectroscopy of bacterial strains and suspensions was performed using a network analyzer in a frequency range from 30 kHz to 6 GHz (HP 8753D) and a coaxial probe (High

In the frequency range between 50 and 300 MHz, dielectric spectroscopy revealed higher

bacterial strains *Micrococcus luteus* and *Bacillus subtilis* compared to the Gram-negative

the same cell count and volume was measured. Particularly at a frequency of 50 MHz

The clear discrimination between the Gram-positive strains *Micrococcus luteus* and *Bacillus subtilis* as well as the Gram-negative strains *Escherichia coli* and *Serratia marcescens* at a frequency up to 100 MHz can be attributed to the β-dispersion. At these frequencies, proteins and other macromolecules of the bacterial cells polarize according to Markx *et al*. [35]. This effect decreases at frequencies above 100 MHz. With increasing frequency the

100

160

spectroscopy, etc.

strains in biological samples [54].

temperature probe). The real

frequency range from 50 MHz to 300 MHz [54].

values of the real part of permittivity (

influence of water becomes more prominent.

strains *Escherichia coli* and *Serratia marcescens* (

(maximum of discrimination), the real part of permittivity

was about 60 units higher than of the Gram-negative strains (Fig. 21)

and imaginary

Using devices with our own configurations, the dielectric properties of different porcine and bovine tissues were determined [25]. Different measuring points were defined on the surface of udder, fat, liver, muscle, and kidney of porcine and bovine tissue (homogenous structure) and the permittivity of these points was measured three times (selected tissues in Fig. 20, left panel). Afterwards, the tissue under these measuring points was excised and dried to calculate the water content. Water content and permittivity ε´ were related to each other, so we could clearly differentiate between fat, low-water-content tissue, with a low permittivity ( 8 ) and liver, muscle or kidney ( 40 ) as high-water-content tissues. The high-water-content tissues show similar permittivity values whereas fat of porcine and bovine origin can be distinguished (Fig. 20, right panel).

**Figure 20.** Determination of the dielectric properties of different porcine and bovine tissues at defined measuring points (left panel). Real and imaginary part of permittivity at 2 GHz of porcine and bovine tissue in relation to the water content (right panel). The standard error represents six independent measurements [25].
