# Morphological Changes in H1299 Human Lung Cancer Cells Following W-Band Millimeter-Wave Irradiation
## 3 Results
### Millimeter Waves Transmit through RPMI 1640 Cell Growth Medium without Generating Thermal Heat  
The wavelength of MMWs is comparable to the layer thickness of the RPMI 1640 medium (2-3 mm) used. Additionally, MMWs undergo multiple reflections in the RPMI 1640 medium. Reflected waves (from the sample) are a combination (interference) of waves reflected from the surface of the nutrition media, the bottom of the dish, and from the cells under investigation. Single (discrete) frequency irradiation regime requires fine adjustments of the distance between the sample and antenna in order to obtain conditions of constructive interference. In contrast, irradiation under a continuous sweeping of frequency allows for repeated wavelength changes over each run, providing conditions of constructive and destructive interference successively. This is advantageous and overcomes the necessity for fine adjustments. Thus, at least during half the duration of a single run, efficient power delivery is obtained. Therefore, all experiments were conducted in the sweeping regime. Although several studies have evaluated the real and simulated dielectric constant of RPMI medium for frequencies up to 72 GHz [17; 18], such data is lacking in the MMW W-band (75-105 GHz) range used in our experiments. Hence, we tested the amount of energy that gets reflected from and penetrates through the culture medium consisting of RPMI 1640 cell growth medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. This basic medium consists of about 40 nutrition ingredients, including various types of proteins present in FBS, diluted in water. The dependence of the reflected and the transmitted signals (normalized to the antenna emitting power P\({}_{0}\approx\) 1 mW) on the thickness of RPMI 1640 medium layer in the Petri dishes was studied for different frequencies (Figure 2a). The ratio of the transmitted and incident power decreases as the thickness of the media increases. For a medium layer thickness of 2 mm, the ratio decreases by a factor of 10\({}^{-4}\)-10\({}^{-5}\). In absolute values, this ratio corresponds to 10-100 nW of power reaching the population of cells adhered at the bottom of the dish. In other words, maximum incident power is absorbed in an RPMI 1640 medium layer thickness of 2 mm. As the medium consists mostly of water, we used the heat capacity of water to estimate the temperature rise during irradiation using:  
\[\Delta T\cong\frac{P_{T}t}{cm} \tag{3}\]  
where \(t\) is the exposure time, \(c\) is the heat capacity (4.18 J/g:K), and \(m\) is the sample mass (1.50 g). This estimation is based on the assumption that the penetrating power decreases exponentially with depth due to an increased absorption of incident energy. Our calculations predict a temperature rise of no more than 0.2-0.4 K for 2-4 min of irradiation of samples immersed in 2 mL of RPMI 1640 medium. Further, the temperature of the medium during the duration of exposure was measured to be in the range of 24-26 \({}^{\circ}\)C.  
In order to maximize the power delivery to cells under irradiation and minimize attenuation loss arising from absorption by culture media, we examined the full frequency spectra (75-105 GHZ) of the transmitted signals penetrating through Petri dishes with different RPMI 1640-layer thicknesses (Figure 2b). Here, \(-7.6\) dB is the free space path loss (FSPL) for the 25-mm separation distance between the two +24 dB gain antennas of our setup, as detailed above (Figure 1). Insertion of an empty dish increased the loss of the initial power by approximately 70% due to attenuation by the polystyrene of the dish (Figure 2b). The transmitted power level in the empty dish decreased by one order of magnitude with the introduction of culture medium, corresponding to an attenuation of about \(-10\) dB. The results indicate an RPMI layer thickness of 0.5 mm as the best working range, translating to a volume of 0.5 mL of RPMI 1640 medium.