Impact of low intensity millimetre waves on cell functions P.H. Siegel and V. Pikov Investigations on the biological impact of low levels of millimetre- wave energy date back to the first experiments on the generation and detection of these high-frequency signals by Sir Jagadis Chunder Bose at the end of the 19th century. Slightly more than a hundred years later, millimetre-wave transmission has become a ubiquitous commercial reality. Despite the widespread use of millimetre-wave transmitters for communications, radar and even non-lethal weapons systems, only a handful of researchers have funded programmes focus- ing on millimetre-wave interactions with biological systems. As such, there is a growing need for a better understanding of the mechanisms of these interactions and their possible adverse and therapeutic impli- cations. Independent of the health impact of long-term exposure to high doses of millimetre-wave energy on whole organisms, there exists the potential for subtle effects on specific tissues or organs which can best be quantified in studies which examine real-time changes in cellular function as energy is applied. In this Letter, a series of experiments are presented which show changes in cell mem- brane potential and the action potential firing rate of cortical neurons under short (1 min) exposures to continuous-wave 60 GHz radiation at mW/cm 2 power levels, more than 1000 times below the US govern- ment maximum permissible exposure. The findings have implications for non-contact stimulation and control of neurologic function, and might prove useful in a variety of health applications from suppression of peripheral neuropathic pain to the treatment of central neurological disorders. Introduction: In 1901 while on a lecture tour in the UK, Sir Jagadis Chunder Bose, the first person to generate, detect and characterise accu- rately millimetre waves, is quoted as stating [1], ‘How lucky we are that the natural eye absorbs this radiation and protects us by veiling our sense against insufferable radiance in these days of space-signaling by Hertzian waves.’ In 1883 the first experiments on free space trans- mission of radio waves for signalling purposes were conducted by Tesla, and in 1884 Bose [2] repeated these experiments using micro- waves. In 1887, Marconi [3] made his famous demonstration of wireless transmission for communications applications at Salisbury Plain, UK. A little more than a hundred years later, we really do find ourselves in a world where we are continuously bathed in low power microwave and millimetre-wave radiation. The widespread deployment of millimetre- wave, and soon submillimetre-wave, generators for wireless telecom- munications [4], airport and checkpoint security screening [5], and even non-lethal crowd control weapons [6], has prompted renewed scientific interest in the effects of this wavelength range on biological materials and organisms. Owing to their established role in the telecom- munications industry, their potential for greater penetration into tissue, and the availability of commercial generators and detectors, millimetre waves have been more widely studied than submillimetre-wave or tera- hertz (THz) frequencies for their biological impact. High levels of milli- metre-wave power absorption have received much attention from the bioelectromagnetics safety standpoint. However, power levels that fall well below the United States Federal Communications Commissions established maximum permissible exposure (MPE) limits of 1 mW/cm 2 for 6 min in the 30–300 GHz frequency regime [7] have received con- siderably less investigation. Neuronal activity is a particularly good marker for gauging stimulus thresholds since the neuronal membrane is optimised for sensing and in conducting electrical impulses with millisecond temporal response. Several research groups [8–18] have noted significant impact on neur- onal activity induced in vivo by modest levels of millimetre-wave exposure (40– 130 GHz, 1 – 100 mW/cm 2 , seconds to minutes) that are not much higher than the MPE. Synchronisation of the firing rate of neurons in the hypothalamus of both rabbit and rat was observed at and below 10 mW/cm 2 [8, 9]. 53 GHz exposure of the sciatic nerve in rats at only 4mW/cm 2 increased the action potential amplitude [10]. Even at 2–3mW/cm 2 , at certain frequencies between 40 and 52 GHz, an isolated frog sciatic nerve showed measureable changes in the amplitude and latency of its compound action potential [11]. Higher levels of millimetre-wave power (10–100mW/cm 2 ), which tend to raise the temperature of the exposed sample, have been shown to produce changes in neuronal activity that sometimes do, and some- times do not, correlate with broadband radiant heating [12–15]. For example, changes in action potential firing rates in skate skin exposed to 130mW/cm 2 at 54 GHz were anti-correlated with those produced by radiant heating [12]. In another study, however, exposure of snail pacemaker neurons to 75 GHz radiation at levels sufficient to raise the temperature several degrees in a few seconds showed changes in firing rate that matched those produced by radiant heating [13]. Similar results, correlating millimetre-wave exposure (62 and 75 GHz) and direct temperature rise, were also reported for the changes induced in ionic currents in these neurons [14]. Most recently, the electrical response of an exposed frog sural nerve (.45mW/cm 2 at 42 GHz) showed threshold effects and transient behaviour that were not well reproduced by broadband radiant heating [15]. Additional studies have focused on millimetre-wave induced changes in cell membrane per- meability. Small increases in current transport across lipid bilayers were seen with both pulsed and continuous-wave (CW) millimetre wave power between 54 and 76 GHz [16]. Similarly, the permeability of phospholipid based liposomes increased after exposure at 130 GHz with 10–17mW/cm 2 [17]. Annexin V, an extracellularly-applied marker, was used to visualise the outward and inward migration of the membrane-forming lipid, phosphatidylserine, during exposure of ker- atinocytes to 42 GHz at 35mW/cm 2 [18]. Radiant heating of the cells failed to reproduce the effect. In a set of experiments on mouse skin receptors, the tail flick response was observed to decrease with milli- metre-wave exposure [19, 20] in contrast to simple heating. A possible explanation for some of these effects may come from recent investi- gations on the prevalence and role of macromolecule-bound water, par- ticularly inside and adjacent to the cellular membrane, which point to strong specific absorption in the millimetre-wave band [21–24]. In our own studies, we have looked at both millimetre-wave induced apoptosis and transient membrane permeability in epithelial cells in vitro [25, 26], as well as real-time changes in the activity and membrane per- meability of individual pyramidal neurons in patch-clamp probed cor- tical slices [27]. The latter experiments were conducted at exposure levels 1000X below the MPE and resulted in at most 38C increase in the tissue bath temperature at the highest exposure level. We believe these experiments yield the strongest evidence to date for significant impact of low-power millimetre waves on cell function. Independent of any human safety related issues, the ability to modulate neuronal activity via optically focused millimetre-wave beams has use as a basic neuroscience tool, and perhaps clinical implications for suppres- sion of peripheral neuropathic pain and treatment of central neurologic disorders. Fig. 1 Top: photograph of single H1299 cell in oxonol and phosphate buf- fered saline, expressing lipid bound fGFP at cell membrane (transfected with pEGFP-F vector) before millimetre-wave exposure. Bottom: same cell after exposure. Red colour indicates FRET induced oxonol fluorescence Experiments on epithelial cells in vitro: In our first set of experiments we employed an immortalised epithelial cell line, H1299 (a gift from Alex Sigel, Caltech, Pasadena, CA, USA) to look at changes in mem- brane permeability with exposure to modest levels of millimetre-wave power: ≏10mW/cm 2 for 2 min at 50 GHz. To enable optical measure- ments of the membrane potential, we transfected cells with a plasmid containing farnesylated green fluorescent protein (fGFP) which binds to the inner leaflet of the plasma membrane. Upon exposure to blue light (490 nm) the fGFP fluoresces green (520 nm), lighting up the inner leaflet (Fig. 1 top). A voltage sensitive dye, oxonol, was added to the media which localises at the outer membrane while the membrane is negatively polarised. The membrane depolarisation evoked by milli- metre-wave exposure causes the oxonol to migrate towards the inner leaflet and become a quenching agent for the GFP fluorescence through a Fo ¨rster resonance energy transfer (FRET) process. The S70 doi: 10.1049/el.2010.8442 Electronics Letters Dec. 2010 – Special Supplement: Terahertz Technology