2.1.

Organism and Culture

The bioluminescent bacterium employed was Vibrio fischeri NRRL-B-11177, kindly supplied by Dr. Michael H. Rayner-Brandes.³⁴ It was cultured at 25°C on agar containing (in g/l): NaCl (Sigma) 30, peptone (Oxoid) 5, meat extract (Oxoid) 3, ${\mathrm{CaCO}}_3$ 5, agar 15, and glycerol 5 ml.³³ After 24-h growth, organisms were washed off the surface of this growth medium using a buffer containing (g/l) NaCl 23, ${\mathrm{Na}}_2{\mathrm{HPO}}_4$ 15.5, nutrient broth (Oxoid) 10, ${\mathrm{NaH}}_2{\mathrm{PO}}_4$ 2, and glycerol 10 ml. Flasks were plugged with cotton wool for aeration and incubated at 24°C overnight for 20 to 24 h. The organisms were then harvested by centrifugation at 3000 rev/min for 10 min using a bench-top centrifuge (MSE Centaur 11). The sedimented bacteria were washed twice using artificial seawater buffer containing (g/l) NaCl 28.0, ${\mathrm{MgCl}}_2·6{\mathrm{H}}_2\mathrm{O}$, 5.0, ${\mathrm{MgSO}}_4·7{\mathrm{H}}_2\mathrm{O}$ 4.5, ${\mathrm{CaCl}}_2·2{\mathrm{H}}_2\mathrm{O}$ 1.5, and KCl 0.8,³⁵ and the same centrifugal procedure. They were finally resuspended in artificial seawater for experiments to a density of $\sim 1\times {10}^8\text{cells}/\mathrm{ml}$. The artificial seawater provides adequate buffering of the cell suspension, ensuring sustained viability, but starves the cells of nutrients. This eliminates any possible increase in light output associated with cell division, which is not permitted under these conditions.

2.2.

TM₀₁₀ Mode Cavity Resonator

The cylindrical resonant cavity used was designed and constructed in-house at Cardiff University’s Centre for High Frequency Engineering from a solid block of aluminum. This block splits into two halves forming a cylindrical cavity of inner radius 46 mm [Fig. 1(a)]. The specific dimensions of this flattened cylindrical device were designed to achieve resonance at 2.45 GHz with a standing wave such that the maximum electric field occurs down its axis and maximum magnetic field at its circumference when resonating in the ${\mathrm{TM}}_{010}$ mode [Fig. 1(b)]. The resonant frequency of the ${\mathrm{TM}}_{010}$ mode is independent of cavity length, but the inner length was chosen to be 40 mm to ensure good spectral separation of the ${\mathrm{TM}}_{010}$ mode from all other modes (e.g., ${\mathrm{TE}}_{111}$) while maintaining a high-quality factor $Q$. This cavity design presents an easily accessed central through-route down its central axis allowing catheter tubing to be inserted vertically, where the E field is at its maximum [Fig. 1(b)]. In the circumferential configuration for H-field exposure [Fig. 1(b)], the tube was held in place by thin strips of double-sided adhesive tape. The tape thickness is very thin ($\sim 100\mu \mathrm{m}$) and was fixed on the outer metal wall, at which point the electric field is reduced to zero. Measurement of the $Q$ factor and resonant frequency with and without the tube and its fixing, which show negligible differences, indicate that the tape has no impact on the E or H field in this position. To allow for the same sample volume ($47.4\mu \mathrm{l}$) for both E- and H-field exposures, an arc length of 50 deg was used for the H-field exposure, meaning that the total length of exposed sample in both cases was 40 mm. Although the heat sinking arrangements are different for the axial and circumferential samples, this is of no issue for comparison between the axial and circumferential experiments since in the former arrangement there is negligible sample heating.

Fig. 1

(a) Inside view of the ${\mathrm{TM}}_{010}$ mode aluminum resonant cavity. (b) Three-dimensional representation of the electric (E) and magnetic (H) field set up, with sample tube placed axially or circumferentially, respectively. E- and H-field intensity plots are also shown.

2.3.

Microwave Hardware and Measurements

The main components of the circuit (Fig. 2) were as follows: (1) microwave signal generator (Telemakus TEG2700-6), which provides a single-tone output at a power level of 6 dBm (i.e., 4 mW rms); (2) microwave switch (Telemakus TES6000-30), which in the “on” position directs the signal to the input of the microwave power amplifier for cavity excitation and into a $50\text{-}\mathrm{\Omega }$ matched load in the “off” position; (3) microwave power amplifier (Mini-circuits ZHL-30W-262) able to deliver a maximum output power of up to 45 dBm (31.6 W rms) and a linear gain of 50 dB; (4) variable attenuator with 30-dB dynamic range; (5) directional coupler (Mini-circuits ZABDC20-322H); (6) circulator with an isolation of 25 dB, and; (7) two average power sensors (Telemakus TED8000-40) measuring incident and reflected power. In order to record any effects without appreciably heating the sample, this system was able to deliver pulsed input power of 26.5 dBm (0.45 W rms), which is considered sufficient to record a measurable response while minimizing the thermal effect.²⁹ It was also powerful enough to produce up to 32-W rms power to explore the response at much higher levels. To do this, a variable attenuator (4) was added to the circuit in order to vary the output power when needed. This required measuring the exact output power before each run using a broadband power sensor (8) (Rhode & Schwarz NRP-Z81). The microwave switch (2) enabled the delivery of power in controlled pulses rather than in a continuous manner in order to avoid heating the sample. We routinely controlled the average power delivered by changing the duty cycle, defined to be % of time during the regular pulsing that the microwave power is switched on. A vector network analyzer (VNA) (Keysight Technologies Fieldfox N9923A) was used to measure and record the reflected power via measurement of the voltage reflection coefficient $S_{11}$, of the cavity to achieve critical coupling of the cavity for maximum power delivery. A circulator (6) was used to protect the power amplifier from the reflected power from the cavity in case there is an unexpected mismatch.

Fig. 2

Microwave circuitry and control system.

2.4.

Software

An in-house LabVIEW program was designed to control the microwave generator and simultaneously record the reflected power (via $S_{11}$) and power delivered to the cavity as the bacteria were subjected to microwave pulses. It also produced a resonant frequency sweep between each treatment. The photon counter (H7467, Hamamatsu) (9) was controlled using a separate LabVIEW program for data acquisition and processing using a 100-ms integration time for all the experiments as this is commensurate with the bacterial response times. This program was also used to plot photon emission rates as a function of time. Each Telemakus device was operated using its own driver, allowing full control over the microwave parameters (e.g., operating frequency and duty cycle). For data synchronization, it was possible to monitor the excitation times by multiplying the integration times by the integrated numbers and visually recording the start and end times for each excitation period. This method proved to be accurate enough for the reaction times that were observed.

2.5.

Experimental Setup

The aim of these experiments was to investigate the biological response of V. fischeri to the separate effects of electric and magnetic fields in the resonant cavity. In the ${\mathrm{TM}}_{010}$ mode, the peak E- and H-field regions are well-separated, and it was, therefore, possible to expose the cells to each field by carefully routing the Tygon sample tube (0.89-mm internal diameter). A precision peristaltic pump (ISMATEC Reglo digital) was used to feed the cell suspension into the cavity through the Tygon tube held in either of the fields within the cavity, and flow was stopped before irradiation. The sample tube was placed in the axial orientation for E-field excitation and circumferential orientation for H-field excitation. The total sample volume in both orientations was $47.4\mu \mathrm{l}$. It was also used to flush the bacterial suspension between runs, thus presenting a new population (from an identical batch) for each run. For microwave H-field exposure at 2.45 GHz, bacterial suspensions were exposed to the maximum available power of 32 W rms and duty cycles of 10%, 20%, 40%, 50%, and 100%. For microwave E-field exposure at 2.45 GHz, bacterial suspensions were exposed to 0.45 or 4.5 W rms at duty cycles of 2%, 5%, and 10%. In all instances, the microwave pulse period was 100 ms and the “off” time varied according to the duty cycle required. The corresponding E- and H-field intensities at the axial circumferential positions are summarized in Table 1. The E and H fields have been calculated using finite-element modeling and these have been shown in our previous paper.²⁹ Such plots show the field distributions, but one still has to perform a calculation to determine the absolute value of the E field on axis, which can then be used to normalize all E- and H-field values at other positions. To do this, we equate the power in to the cavity to the power dissipated in the suspension, and in doing so we end up with the E-field normalizing factor used in Table 1. The specific absorption rate (SAR) for the E-field excitation is 4.5 W dissipated in a sample volume of $47.4\mu \mathrm{l}$, which equates to 95 kW/kg. However, this is the peak SAR and, since we pulse our measurements, multiplication of this figure by the associated duty cycle is required to determine the time averaged SAR. Although the heat sinking arrangements are different for the axial and circumferential samples, this is of no issue for temperature rise since in the former arrangement there is negligible sample heating.

Table 1

Electric (E)- and magnetic (H)-field levels at 2.45 GHz averaged across the entire bacterial suspension within the TM010 mode cavity when the sample tube is placed separately in the axial or circumferential positions. The first the sample was tested in the E field (axial) and then a different sample, containing the same density and volume of bacteria in artificial seawater buffer, was tested in the H-field (circumferential) orientation, with critical coupling achieved for each measurement.

2.6.

Cavity Coupling

All experiments described in this paper used the lowest resonant mode ${\mathrm{TM}}_{010}$ of the other higher order resonant modes which co-exist. Depending on the dielectric properties of the material placed within the cavity, the resonant frequency and $Q$ factor of the cavity may be altered due to associated changes in the energy stored and energy dissipated, respectively. These changes are quantified by “perturbation theory,” which is based on the assumption that the actual fields of a material-filled cavity are not greatly different from those of the unperturbed cavity.³⁵ A high $Q$ means low losses, and vice versa. The $Q$ factor of the sample-filled cavity is determined by the conductor losses in the cavity walls and the dielectric losses in the sample, the latter usually being dominant for aqueous samples. For the experiments presented here, $Q$ factors were calculated from data collected using a VNA by measuring the reflected power $S_{11}$ against frequency and subsequent curve fitting to obtain $Q$, resonant frequency, and coupling coefficient $g$ to the cavity; this latter (dimensionless) quantity is defined via the input impedance of the cavity at resonance, which can be written $Z_{\text{in}}=g{Z}_0$, where $Z_0$ is the system impedance, in our case $50\mathrm{\Omega }$. Curve fitting to the measured $|S_{11}|$ in the frequency domain yields the loaded $Q$ factor ($Q_L$, i.e., including the effects of the coupling structure), coupling coefficient $g$, and resonant frequency $f_0$. The unloaded $Q$ factor ($Q_U$) is simply related to the loaded $Q$ via $Q_U=(1+g)Q_L$, and this value is used to estimate the peak E and H fields within the cavity. All microwave connections are via coaxial lines of characteristic impedance $50\mathrm{\Omega }$. The actual coupling structure is a loop, which terminates a microwave $N$ connector, which couples to the high H field of the ${\mathrm{TM}}_{010}$ mode close to its perimeter. The loop can be rotated, allowing fine adjustment of the coupling coefficient $g$. The cavity is said to be critically coupled when maximum power transfer between the resonator and the feedline is achieved ($|S_{11}|=0$). This is when the device is impedance matched to the input circuitry at the resonant frequency, i.e., when $g=1$. The goal is, therefore, to obtain a value of $g$ as close to 1 as possible by fine initial adjustment of the orientation of the coupling loop, in order to ensure maximum power transfer to the cavity, and so to utilize effectively all of the microwave input power in generating high electromagnetic field amplitudes within the cavity. When critical coupling is attained, $S_{11}$ tends to zero, giving a pronounced “dip (in dB scale)” when measured using the network analyzer. In practice, values of $|S_{11}|$ of $<0.05$ are achieved, giving a measured insertion loss of less than $-25\mathrm{dB}$ at resonance.

2.7.

Photon Counting

The photomultiplier of an integrated photon counting device (Hamamatsu H7467 series) was powered from a stabilized 5 V DC supply (Keysight Technologies) and optically coupled through a fiber-optic cable to the catheter tube containing the bacterial suspension delivered for microwave component field exposure (a selected volume of 47.4 ml of the pumped stream). This detector has a spectral response between 300 and 650 nm with a peak sensitivity wavelength of 420 nm that makes it ideal for its use with organisms exhibiting maximal bioluminescence at around 490 nm. The photomultiplier integration time was 100 ms. Due to its high sensitivity, it was essential to rigorously isolate the detector from any ambient light to avoid any light leaks and ensure that it was stored for at least 30 min in total darkness before use in order to reduce background dark current to a minimum and signal-to-noise ratio to a maximum.

2.8.

Temperature Measurements

An infrared thermal imaging camera (Micro-Epsilon thermoIMAGER TIM 640) was used to measure temperature changes during experiments. The camera was carefully focused on the sample tube through a side port in the cavity and is sensitive to within 1°C, has a range of $-20°\mathrm{C}$ to $+900°\mathrm{C}$ at ambient temperature (0°C to 50°C) and an acquisition rate of 30 ms. All temperature values quoted represent the maximum temperature measured across the sample (5 mm). Note that the temperature measurements overshoot the microwave excitation period due to the lag between the temperature of the cell suspension and that of the sample tube (the thermal imaging camera being focused on the outer surface of the latter).

3.1.

Critical Coupling

Resonant traces for $S_{11}$ are shown in Fig. 3 and accompanying coupling parameters given in Table 2, for the E-field excitation configuration either with the cell suspension in artificial seawater or for an empty cavity. The high losses incurred when the seawater sample is present can be seen in Fig. 3(a). This affects the critical coupling condition as shown, so further adjustments of the coupling are required to ensure maximum power transfer. Also listed in the associated table are the resonant parameters of resonant frequency, $Q$ factors, and coupling coefficient $g$, linked to the traces shown. Similar traces are shown in Fig. 3(b), this time for the H-field configuration. In this configuration, it is observed that the loss caused by the presence of the cell suspension is hardly detectable and a high $Q$ factor is maintained, even when the aqueous sample is present. In either sample position, we do not plot the control data for a blank sea water sample as, within experimental error, the sea water trace is identical to that for a bacterial sample; this is to be expected, since the bacteria occupy a small volume fraction ($<0.1\%$) and have dielectric properties close to sea water.

Fig. 3

(a) The magnitude of the cavity’s amplitude reflection coefficient $S_{11}$ in the frequency domain for the the E-field configuration and (b) H-field configuration, with (continuous trace) and without (dashed trace) cell suspension. Note the profound difference in frequency scale between (a) and (b).

Table 2

Calculated values for the loaded quality factor (QL), the coupling coefficient g, and unloaded quality factor (QU), together with the TM010 resonant frequency f0, derived from the data presented in Figs. 3(a) and 3(b) for the E and H fields. All measurements were taken at low power (1 mW).

3.2.

Background Bioluminescence

We measure “dark” background photon counts, which are acceptably low, $<4\text{photons}/\mathrm{s}$ obtained with 47.4 ml of artificial sea water in the absence of bacteria in the Tygon tube within the cavity. Similar results were obtained irrespective of whether the E- or H-field configurations were employed provided that the photomultiplier was always completely shielded from ambient light and the photocathode was stored and equilibrated under these conditions. It was also necessary to determine the decay of light emitted from the bacteria in their starved condition in seawater. Prior to microwave exposure, all bacterial samples are thermally equilibrated in the cavity for around 30 min. Typical microwave exposure occurs over a timescale of around 300 s. The time constant of the natural decay of bioluminescent light output is $\sim 2$ days, so over a typical experiment there would be a natural decay of around 0.2%, which is negligible compared the effect of small thermal fluctuations (of the order of 0.1°C).

3.3.

Magnetic (H) Field Effects on Bacterial Bioluminescence

V. fischeri cell suspensions were subjected to pulsed H field for 10 s (between $t=10\mathrm{s}$ and $t=20\mathrm{s}$) as shown in Fig. 4. Different duty cycles were tested, ranging from 10% to 100% for an output power of 32 W rms, resulting in a maximal H-field strength of 183 A/m. In all cases, there was no noticeable variation in light emission during or after H-field excitation of the cells.

Fig. 4

The response of Vibrio fischeri light output to magnetic field excitation at 32 W rms and duty cycles of 10%, 20%, 40%, 50%, and 100%. The time duration of exposure is indicated by the black bars on the $x$ axis. These traces are representative of at least two independent experiments.

3.4.

Electric (E)-Field Effects on Bacterial Bioluminescence

Light emission from suspensions of V. fischeri responded to microwave E-field pulses at all power levels and duty cycles tested. To investigate the sensitivity of bacterial bioluminescence to microwave E-field exposure and examine whether the effect is thermal or nonthermal in origin, simultaneous temperature measurements were conducted alongside photon counts over a range of duty cycles: 2% [Figs. 5(a) and 5(b)], 5% [Figs. 5(c) and 5(d)], and 10% [Figs. 5(e) and 5(f)], and at 0.45 W rms [Figs. 5(a), 5(c), and 5(e)] and 4.5 W rms [Figs. 5(b), 5(d), and 5(f)]. At 0.45 W rms, equivalent to an E-field intensity of 4.24 kV/m, temperature increases of 0.6°C, 1°C, and 1.5°C (at rates of 0.02, 0.03, and 0.05°C/s) were observed for duty cycles of 2%, 5%, and 10% [Figs. 5(a), 5(c), and 5(e)], respectively. This was accompanied by a 10% enhancement in light output for all three duty cycles tested, at increasing photon emission rates of 0.4, 0.6, and 0.7%/s, respectively, for each duty cycle. In the case of 10% duty cycle [Fig. 5(e)], this initial enhancement of bioluminescence was followed by suppression in light output between 16 and 30 s E-field exposures (a reduction of 10%). At the end of the 30-s treatment time, there was an immediate recovery in light output (15% enhancement) at a rate of 0.8%/s, after which a steady-state was achieved, and light output followed a natural decay curve (the latter also being the case for 2% and 5% duty cycle treatments).

Fig. 5

(a) Simultaneous monitoring of Vibrio fischeri light output and temperature change under microwave E-field stimulation at 0.45 and 4.5 W rms and (a) and (b) 2%, (c) and (d) 5%, and (e) and (f) 10% duty cycle. The time duration of exposure is indicated by the shaded box. The exposure time for (a)–(e) was 30 s, and that of (f) was 15 s, due to the fact that microwave E-field excitation beyond this point resulted in complete inhibition of cells (i.e., no appreciable recovery of light emission). All experiments were repeated at least in duplicate. The $y$ axis of (a)–(f) represents the photon counts (i.e., bacterial bioluminescence, $B$) normalized to the steady-state baseline level ($B_0$) before E-field exposure. The baseline photon counts before microwave exposure for each figure were as follows: (a) 538, (b) 1464, (c) 602, (d) 1006, (e) 422, and (f) 782 photons/s. (g) A summary of the change in light output achieved during the inhibitory and recovery phases during or immediately following E-field pulses at 4.5 W rms and 2%, 5%, and 10% duty cycles. The $y$ axis of (g) represents the change in photon counts, based on data derived from (b), (d), and (f). Error bars represent the standard deviation, $n\ge 6$ successive pulses.

At 10 times higher power (4.5 W rms, equivalent to 13.4 kV/m), temperature increases of 3.2, 5.4, and 6.3°C/s (at rates of 0.11, 0.18, and 0.21°C/s) were observed for duty cycles of 2%, 5%, and 10% [Figs. 5(b), 5(d), and 5(f)], respectively. In all cases, bacterial bioluminescence responded quickly (within the 100-ms acquisition time) and sensitively to the 100-ms microwave E-field pulses. At duty cycles of 2%, 5%, and 10%, the average rate of suppression in bacterial light output during every 100 ms E-field pulse was 0.57 ($\pm 0.31$), 0.94 ($\pm 0.58$), and 0.59 $(\pm 0.50)\%/\mathrm{s}$, respectively. Note that the total excitation time was 30 s in all experiments, apart from that of 10% duty cycle, which was 15 s, due to the high degree of inhibition observed over this exposure time. There was no significant difference between the inhibition and recovery rates during or after the microwave E-field-pulses at all duty cycles tested. Likewise, there was no significant difference between the percentage changes in bioluminescence during these inhibitory and recovery phases, when all data was pooled together [Fig. 5(g)]. However, there was a general trend of increasing stimulatory and inhibitory rates from pulse 1 to 6, followed by a decrease in these rates from pulse 6 to 12, as light levels approached zero. Over the total exposure time, the overall rate of inhibition of bacterial bioluminescence was 3.1, 3.1, and 5.8%/s. The overall rate of recovery of bacterial bioluminescence following exposure was 6.1, 0.5, and 0.1%/s, respectively, with steady-state established at 80%, 40%, and 20% of maximum light output levels.

3.5.

Thermal Effects

Temperature increases were measured in all experiments where bacteria were exposed to microwave E field. The rate of temperature increase was proportional to the amount of energy (i.e., power and duty cycle) delivered into the cell suspension. The maximum temperature achieved was 32.2°C ($\mathrm{\Delta }T=6.3°\mathrm{C}$ at 10% duty cycle, 4.5 W rms, 15 s), which caused a significant decrease in light output, down to 3% of the maximum (i.e., a 97% decrease). The minimum increase in temperature observed was 0.6°C (2% duty cycle, 0.45 W rms). All temperature measurements are recorded in Table 3 and Figs. 5(a)–5(f).

Table 3

A summary of the E-field (2.45 GHz) treatment regime, including the pulse period, recovery time, total exposure time, number of pulses, as well as the temperature elevations (temperature increase, ΔT and maximum temperature achieved, Tmax) for 2%, 5%, and 10% duty cycle.

In order to examine if the microwave E-field-induced effects on bacterial light output were entirely thermal in origin, or if there may be an “added effect” induced by E-field exposure, a conventional heating control was conducted for comparison. Figure 6 shows the effect of water bath heating to 31°C on bacterial light output. The results can be compared directly to the bacterial suspension that was exposed to 4.5 W rms, 5% duty cycle for 30 s, as the same maximum temperature of 31°C was achieved ($\mathrm{\Delta }T=5.4°\mathrm{C}$ for E-field exposure and 5.8°C for conventional heating). Water bath heating led to a maximum inhibition in light output of 15%, and returned to the baseline level after heating. In contrast, E-field excitation resulted in reduction of light output to 4%, which recovered to only 40% after exposure. It should be noted, however, that the rate of heating for E-field excitation was $\sim 4$ times faster for E-field excitation (0.15°C/s) than was the case for conventional heating (0.04°C/s).

Fig. 6

Simultaneous monitoring of Vibrio fischeri light output and temperature change under conventional heating using a water bath. The time duration of heating is indicated by the black bar on the $x$ axis.