It has been known for more than a century that living cells emit ultra-weak light (UWL) or photons [JJC13, DUI97] – a phenomenon also refered to as bio-photons. However, it is still not clear what role these bio-photons play nor how they are generated in the first place [CP14]. Although the exact process responsible for the bio-photons has not been identified, there is a strong indication that metabolic processes are involved [ZTZ73]. It is tacitly assumed that bio-photons originate from radiative decay of electronically excited molecular species, which are generated chemically during oxidative metabolic processes in the mitochondria of microbial, plant and animal cells. These molecules, known as Reactive Oxygen Species (ROS), have a number of variants with multiple mechanisms for their pro- ductions [PPR14]. Among these variants are the triplet excited carbonyls (emitting mostly in the visible part of the spectrum, 350–550 nm), dimolar (emitting at around 634 nm and 703 nm) and monomolar singlet oxygen (emitting at 1270 nm) [PPR14]. The mechanisms underlying the generation of ROS can be classified as occurring either during the normal functioning of the cell (i.e., the electron transport chain) or initiated by various biotic or abiotic stressors and oxidative factors (i.e., chemically induced oxidative stress) [PP11]. It is important to note that other forms of bio-luminescence, such as fluorescence or phosphorescence, also occur in living organisms. What distinguishes bio-photons as described above is the spontaneous nature of their emission.
It is possible that bio-photons are merely a byproduct of such metabolic activity in the cells. However, a number of studies point to a functional role of the bio-photons within organisms, from the control of embryonic development [Gur23, GA28, VB15] to information transmission in the nervous system [BSTA10, SVB+15, ZKT+18, GKK04, RTB+11]. A recent study found that light can trigger a physiologically meaningful function (i.e., thermogenesis) deep in the brain (via the light sensitive protein opsin5) [ZDU+20]. A number of experiments have established a correlation between the intensity of the bio-photons and the firmly accepted electro-chemical signalling of the neurons [TD14]. In particular, the application of glutamate, which is the most abundant neurotransmitter in the brain, has shown to significantly enhance the bio-photon emission in mouse brain slices while certain action potential inhibitors (e.g. Tetrodotoxin) or anesthetics (e.g. procaine) suppressed the bio-photon emission. In other studies, the electroencephalographic (EEG) activity from the brain was found to be strongly correlated with the emission intensity of the bio-photons [KTS+99, DSP12]. Such results manifest a potential connection between brain activity and bio-photons generation, but for an indication of a functional role we look at the capability of neurons to guide light and, thus, their potential for being involved in neuronal communication [SVB+15]. Note that even if the observed bio-photon intensity is weak and, thus, seemingly ineffective as a signal carrier, the intensity may be much larger inside cells [BSTA10]. The light guiding capacity of neurons has been shown both in models and experiments [SWD10, HMB+94, DGB+20, KBT+16]. The first direct experimental evidence obtained using rat spinal nerve roots suggested that light stimulation can generate bio-photons, which are transmitted through the neural fibers [SWD10]. In addition to this, multiple experiments have shown a directional dependence for light propagation in the neural tissue; including the observation of increased transmission along the axes of the white matter tracts [HMB+94] and a recent study in which the scattering coefficients of the white matter in spinal cord of various species were found to be significantly lower in the longitudinal direction i.e., along the axis of the axons [DGB+20]. These observations spurred theoretical modelling of the light guiding properties of axons. A detailed model based on photonic simulation software found that myelinated axons, i.e. axons enclosed in a myelin sheath, are capable of guiding a large range of wavelengths over distances relevant for connecting separate parts of a brain [KBT+16].
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The observations of bio-photons to date have generally seen a very low count of about 1-100 photons per second [CP14]. Hence, the main technical challenge of detecting bio-photons is that it requires detectors with high efficiency and low false counts, which mask the bio-photons signal. To achieve such high signal and low noise detection, most previous studies have utilized either photo-multiplier tubes or silicon based Charged-Coupled Device (CCD) based imaging, both of which have some limitations [CP14]. Photo-multiplier tubes are robust detection devices for low light-levels due to their low dark-current. However, they provide almost no spatial resolution and have maximum photon detection efficiencies of about 25-30% over approximately a 100 nm range [HC07]. In contrast CCD devices allow imaging samples and thus provide spatial resolution. Their peak sensitivity can reach above 80%, and with an electron-multiplier enhancement they can reach single-photon detection sensitivities. However, for all but the most advanced devices, the signal-to-noise ratio for single-photon detection is insufficient for bio-photon detection [CP14]. Another key limitation is that CCD devices, due to the value of the energy band-gap of silicon, are insensitive to radiation above about 1000 nm, and thus the potential wavelength of some of the bio-photons, e.g. corresponding to the decay of monomolar singlet oxygen.
To overcome the limitations of previous approaches, we use a photon-detection technique based on superconducting nano-wire single-photon detectors (SNSPDs). The development of this technology was driven by similar requirements for efficient single-photon level detection in quantum communication [HI19, You20]. The current state-of-the-art SNSPDs have close to unity efficiency (> 95%) [MVS+13] and less than one erroneous (dark) count per second [SSTT15, SVD+15]. For this reason, they are ideally suited for bio-photon detection. In addition SNSPDs feature unprecedented timing resolution of a few pico-seconds [KZA+20], and can be tailored to operate over a wavelength range – from the ultraviolet to the infrared – which is broader than the standard detector platforms, i.e. photo-multipliers and CCDs. The fact that SNSPDs, which have to be placed in a cryogenic environment, are interfaced using optical fibres facilitates spatial resolution of the bio-photon emission and thus allow identification of regions in the neuronal tissue with increased or reduced rates of bio-photon emission.
Bio-photon emission has been studied in a range of biological systems from plants to neural tissue of mammals. Indeed, most thorough studies have employed brain or spinal-cord tissue from rats. We use tadpoles from the frog Xenopus as our model. This system has a high degree of conservation of most essential cellular and molecular mechanisms as compared to mammals and it is inexpensive and easy to manipulate genetically. Moreover, Xenopus has been used to determine the molecular mechanisms that control neuronal differentiation and network organization of light sensory circuits in the retina [BHM14, BM18, BALM+20]. Such studies are relevant to the role of light sensitive proteins, such as opsin5, which was found facilitate light induced functions in the hypothalamic preoptic area of a mouse brain [ZDU+20]. Finally, the relatively fast development of tadpoles makes it possible to observe embriogenesis from a single-cell embryo to a fully developed tadpole brain over a span of only four days. Thereby, this allows correlating bio-photon emission with specific developmental stages from undifferentiated embryonic cells (day 1), brain progenitor cells of the neurula (2 days) and differentiated brain cells (day 4). During this period of embryonic development, neural circuits begin to wire (second day, initial neuron differentiation) to form a complex network by day four.