Some of the abstracts were published at http://ebook.journalams.com/201310/EBook.htm
Biophoton imaging technology and its application.
Ultraweak photon emission from living organisms, biophoton, has long-time been expected to use as a signal mediator carrying pathophysiological information of living body. Because of its potential in non-invasiveness being applicable for a wide variety of field covering from agriculture to medicine, biophoton has been very attractive as providing an ‘ultimate’ methodology for diagnosis. However, from the discovery of biophoton emission, more than half a century has passed. Although a number of phenomena have been revealed, practical application of biophoton has still been restricted owing to the weakness. To characterize biophoton emission in detail and extract worthful information; spectral, spatial, and other optical properties, or all of them, should be considered. Development of technology to analyze optical properties under the condition in such a single photon level is crucial. From 1970s, in Japan, Professor Inaba and his projects leaded the field of biophoton research as one of the pioneers and achieved various results from measuring technology to the mechanisms. In my talk, I will review the history of biophoton study along with technology development, particularly focusing to biophoton imaging, and discuss what was achieved and what is necessary for further advancement of biophoton analysis for practical application as a diagnostic tool.
Mechanistic aspects on the formation of electronically excited species by metabolic oxidative process – role of reactive oxygen species
Pavel Pospíšil, Ankush Prasad, Marek Rác and Anshu Rastogi
It is well known that biological systems such as microorganisms, plants and animals including human beings forms spontaneously electronically excited species by oxidative metabolic processes. In spite of the fact that the mechanism responsible for the formation of electronically excited species is not clearly understood, several lines of evidence have been provided that reactive oxygen species (ROS) are involved in the formation of electronically excited species. The review attempts to describe the role of ROS in the formation of electronically-excited species as detected by ultra-weak photon emission. It is summarized that the oxidation of biomolecules such as lipids, proteins and nuclei acids by ROS incites a cascade of reactions leading to the formation of triplet excited carbonyls formed by the decomposition of unstable intermediates such as cyclic endoperoxide (dioxetane) and linear tetroxide. When chromophores are in the close proximity of triplet excited carbonyls, the triplet-triplet energy transfer from triplet excited carbonyls to chromophores results in the formation of excited chromophores. Alternatively, when molecular oxygen is present, the triplet-singlet energy transfer from triplet excited carbonyls to molecular oxygen brings about the formation of singlet oxygen. The understanding of the mechanistic principles on the formation of electronically-excited species allows us to use ultra-weak photon emission as a non-invasive tool for study of the oxidative metabolic process in the cells.
Mechanism of light-induced ultra-weak photon emission via photosensitization reaction
Ankush Prasad and Pavel Pospíšil
The solar radiation comprising of visible light and ultra-violet (UV) radiation have severe negative impacts on the organism. The radiations are known to initiate the formation of reactive oxygen species (ROS) by photosensitization reactions (type I and type II). In the photosensitization reaction, the absorption of excitation energy by photosensitizer causes the transition from the ground state to the singlet excited state, which is later converted to the triplet excited states via intersystem crossing. The ROS formed by both Type I (superoxide anion radical,O2·-; hydrogen peroxide, H2O2; hydroxyl radical, HO·) and Type II (singlet oxygen, 1O2) photosensitization reactions have a capability to oxidize biomolecules such as lipids, proteins and nucleic acids forming electronically excited species finally leading to ultra-weak photon emission. The presentation will deal with the results on one- and two-dimensional spontaneous and induced (visible light and UVA radiation) ultra-weak photon emission imaging. The correlation of ROS and ultra-weak photon emission will be supported by experimental results performed on different models to decipher the processes involved. The mechanism of photosensitization reaction leading to ultra-weak photon emission will be presented.
Experimental evidence on the role of biomolecules in the UPE
Marek Rác, Pavel Pospíšil
Despite the fact that the phenomenon of UPE is already known for almost a century the mechanisms are still not fully examined. Oxidative damage on human U937 leukemia cell culture was detected by ultra-weak photon emission, high-performance liquid chromatography (HPLC), dot blot and comet assay as well as UPE from isolated biomolecules was studied in order to provide experimental evidence supporting the current hypothesis.
Evidence that hydrogen peroxide (H2O2) and hydroxyl radical (HO·) catalyze oxidative damage in human U937 leukemia cells will be presented. Detection of malondialdehyde (MDA), byproduct of lipid peroxidation, by isocratic reverse-phase HPLC revels HO· caused lipid peroxidation while H2O2 caused no effect after 30 min. incubation. Dot blot reveals that HO· caused much higher oxidative damage to proteins that H2O2 while thanks to comet assay it can be said that there was some but no significant difference in oxidative damage of DNA caused by H2O2 and HO·.
Based on those results we can conclude that in the case of H2O2 induced UPE from human U937 leukemia cell culture proteins were the major source of the UPE. Also data obtained by measurement of the induced UPE from isolated biomacromolecules show that proteins are more likely initial target of H2O2.
Lipid hydroperoxides and singlet molecular oxygen generation: an update
Sayuri Miyamoto and Paolo Di Mascio
Lipid hydroperoxides (LOOH) including fatty acid, phospholipid, cholesterol and cholesteryl ester hydroperoxides have been detected and characterized both in vitro and in vivo. These hydroperoxides are formed by enzymatic and non-enzymatic mechanisms. Under normal conditions, LOOH are converted to the corresponding hydroxides by antioxidant enzymes. However, there are situations where LOOH are not completely reduced and undergo reactions to generate alkoxyl and peroxyl radicals. In this context, our group has characterized in details the generation of singlet molecular oxygen (1O2) from reactions involving LOOH and biologically relevant oxidants, such as, metal ions, peoxynitrite, HOCl and cytochrome c. The generation of 1O2 was directly evidenced by spectroscopic detection and characterization of its light emission at 1270 nm. Moreover, using 18-oxygen labeled hydroperoxides we detected the formation of 18O-labeled 1O2 by chemical trapping with anthracene derivatives and LC-MS/MS detection of the corresponding labeled endoperoxides. Using this approach we could clearly demonstrate that LOOH either in the form of free fatty acid hydroperoxides or in membranes can undergo reactions leading to 1O2 generation. More recently, we have also identified the generation of 1O2 in a more complex system involving cardiolipin-cytochrome c interaction. Interestingly, using this model system we were able to detect a continuous generation of 1O2 over the course of cytochrome c-promoted cardiolipin oxidation pointing to the potential generation of 1O2 during mitocondrial metabolism.
Seedlings’ stress and rhythms in UPE experiments
Thiago A. Moraes, Eduardo Bertogna and Cristiano M. Gallep
The time pattern of seedlings’ biophotons appears in tune with the local gravimetric profiles, as recently shown for wheat samples. Effects due transcontinental acclimatization found in recent, long term experimental series simultaneously run in Brazil, Czech Rep. and Japan are presented, as well as experiments with single (sunflower) seedlings. Discussion on recent data and proposal for future intercontinental experiments will be fomented, since many opportunities in chronobiology and physiology are open.
Also, data of tests for control and toxic samples will be presented. Simultaneous procedures are strictly necessary in the case of germination tests to be properly compared in terms of spontaneous biophoton emission, since circadian rhythms are always present, as already shown ,,. New series of germination tests using NaF (EC50, 50mM) are presented, with clear discrimination of the stressed, abnormal UPE emission in less than 12 hours. By simple local linear fitting, it was possible to distinguish stress from control samples with 95% confidence
Delayed Luminescence spectroscopy to monitor mitocondrially targeted effects of cell proliferation inhibitors
In order to gain new insights into the biochemical mechanisms responsible for the low-level photoinduced Delayed Luminescence (DL) of living cells, as well as to provide new data regarding the relation between DL and the cell status, a new set of experimental data was performed. In this study the relation between DL induced by UV-laser excitation and the effects of agents that inhibit cell proliferation and induce apoptosis in various cancer cell types were investigated.
Delayed Luminescence measurements from liquid suspension of cultured cells, in the time interval 10 μs – 10 ms after the switching off of the illumination source, were performed on treated samples and compared to untreated ones.
The obtained data showed to be consistent with an important role of the Mitochondrial Respiratory Chain in DL. In particular the role of MRC Complex I in DL of human leukemia Jurkat T cells was probed by using Complex I targeting agents like rotenone, menadione and quercetin. In addition, the effects of berberine, that is known to be selectively accumulated by mitochondria and slow mitochondrial respiration, on follicular and anaplastic thyroid cancer cells were studied, showing a different spectral DL response in the two different cell types. Moreover results show the possibility of using Delayed Luminescence to monitor the effects of anticancer treatments.
Variable magnetic field influence on leukocytes – the analysis on the base of luminol chemiluminescence
Honorata Nawrocka-Bogusz, Michal Cifra, Barbara Poniedziałek
In this study we attempted to assess the effect of the variable magnetic fields on reactive oxygen species (ROS) production in leukocytes of human blood (mainly monocytes and neutrophils) in vitro.
Neutrophils are phagocytic cells. As well as lymphocytes and macrophages they are involved in the effector immune response. They are so-called ”rapid response forces”. Their basic function is the destruction of pathogens (bacteria, viruses, fungi) and phagocytosis of microorganisms.
Monocytes form heterogeneous cell population. On the one hand they are a transitional stage of tissue macrophages and dendritic cells; on the other hand they part of the immune response. Monocytes migrate to places of ongoing inflammation. As well as neutrophils they represent the second cells migrant population. They are not only capable of phagocytosis and killing of microorganisms, but also to the memory-antigen presentation to lymphocytes T.
Neutrophils and monocytes destroy harmful microorganisms by the mechanism called “oxidative burst” (respiratory burst). During the oxidative burst the reactive oxygen species (ROS) and reactive nitrogen species (RNS) such as O2·, H2O2, HOCl, NO, NO2, NH2Cl are produced.
Blood samples from healthy blood donors were used for the study. Lithium heparin was used as the anticoagulant. Neutrophils and monocytes were isolated from full human blood by using density gradient method. The respiratory burst was induced with PMA (phorbol 12-myristate 13-acetate) in order to produce submaximal stimulation of the respiratory burst.
Luminol was used as a luminescent probe for ROS generated by neutrophils and monocytes during their oxidative burst. For other parameters being the same, the higher is the concentration of oxygen species the higher is the detected luminol chemiluminescence.
The variable magnetic field of ELF range of the time averaged induction of 44.5 µT was applied for 30 minutes in the dark at room temperature. The level of the magnetic induction application was growing at 12 seconds interval starting from 4.45 µT to the selected, then the intensity drops to the initial value, the process is repeated cyclically.
Luminol chemiluminescence has been measured using a selected low-noise photomultiplier module H7360-01 in light tight chamber.
We observed that variable magnetic field changed the activity of neutrophiles and monocytes during the respiratory burst. This was seen on the base of the intensity of chemiluminescence. The kinetics of the of the chemiluminescence due to oxidative burst process differed between magnetic field treated and non-treated leukocytes. We observed tendency of the luminol photon emission to the decreased after magnetic field exposition.
Review of Ultraweak Photon Emission (UPE) by Different Living Systems and Experimental Methods of UPE Detection
Vahid Salari, Christian Brouder
Here, we will review ultraweak photon emission (UPE) by different types of biological systems (e.g. plants, bacteria, human body, etc ) and their likely sources of photon emission mainly based on their spectrum. Different methods of experimental detection of UPE will be reviewed as well.
Ultra-weak photon emission as possible mechanism of cell-cell interaction
Claudio Rossi, Agnese Magnani and Stefania Lamponi
The fundamental prerogative of Life is communication. Living organisms and their components are open systems far from equilibrium, in which matter, energy and information are exchanged within specific and coherent ranges. The asymmetrical nature of time for organisms, and indeed any system, is generated by a complex network of interactions, which are irreversible in nature. This complex network of interactions describes how individuals and their own systems connect to each other by processing information arising from the exchange of different kinds of signals. The complex nature of signalling is still to be exhaustively explored (in particular the “non-chemical” signalling) and it is likely that this complexity increased during evolution. Chemical signalling has been profoundly investigated and many mechanisms of cell-cell chemical interaction are well established. On the contrary, physically mediated interactions in living organisms are poorly understood, especially those supported by electromagnetic, signalling. In order to explore if electromagnetic radiation is involved in modulating cell processes during cell development we studied the cells behaviour during their growth. In particular in this contribution we would present our experimental results designed to evaluate whether different cell populations physically interfere when incubated in separate Petri dishes placed in close proximity. In our experiments two different cell populations, immortalized mouse fibroblasts (NIH3T3) and adult human microvascular endothelial cells (HMVECad) were selected and seeded in separate polystyrene Petri dishes. Dishes were then placed one on the top of the other at a distance of 4mm. Cell number and morphology of NIH3T3 and endothelial cells were found to be modified suggesting that specific signalling were transmitted through the polystyrene wall affecting cell proliferation rate and morphology. We also investigated if the observed morphological changes of the cell systems were also accompanied by a modulation of the cell metabolites. NMR spectra were then collected on “in vivo” cell-cell systems to explore the differences of the cell meatabolic profile induced by electromagnetic signalling.
Radiation-based cell communication
The presentation starts with a short introduction into cellular fields due to ions, polar molecules and radical reactions. Even though this leads to frequencies from radio waves to UV-light, the basic hypothesis for radiation-based cell communication assumes visible and UV-light as the major frequency band.
The major focus will be on endogenous radiation used for cell communication and the barrier-method as a tool for isolating such radiation from chemical signals. We further classify into radiation-based (i) induction of chemical reactions and (ii) effects on cell division rates (i.e., radiation-based quorum sensing).
A third part refers to the general sensitivity of cells to radiation from the environment, hence not only to those from neighbor cells, but as well from technical devices or the solar activity.
Two hypothetical examples illustrate, further, the potential of applied research regarding non-invasive technology.
The major conclusion will be that the molecular structures of cells generate electro-dynamic fields (signals) that feed back on cells and, therefore, play an essential role in the self-organization of life.
Evidence for non-chemical, non-electrical intercellular signaling
Synchrony between physically separate biological systems is well known. We posed the question: Can cells induce synchronous behavior in neighboring cells which are physically separated and which therefore cannot communicate via chemical or electrical mechanisms. Caco-2 cell line cultures were divided into three groups. “Inducer” cells were exposed to H2O2. “Detector” cells were placed in separate containers near the inducer cells but were not exposed to H2O2. Control cells were exposed to fresh media and were kept in a distant lab area. Samples were measured for total protein concentration, NF-κB activation and structural changes, 10, 30 and 60 minutes after exposure respectively. Here we show exposing inducer cells to H2O2 result in a significant reduction in total protein content (-50%), an increase in nuclear NF-κB activation (+38%), and structural damage (in 56% of cells) compared to controls. There was a similar reduction in total protein content (-48%), increase in the nuclear fraction of NF-κB (+35%) and structural damage (in 25% of cells) in detector cells. These findings provide evidence in support of an NCNE communication between intestinal cells. This signaling system possibly plays a role in synchronous, stimulus-appropriate cell responses to noxious stimuli. The nature of this signaling system is unknown. However, biophotons are appealing candidates. This phenomenon may explain a number of cellular behaviors that are hard to explain based only on conventional cell signaling systems.
Optical interaction of tissue cultures
Kateřina Červinková, Anshu Rastogi, Michal Cifra
This study is focused on the topic of optical interaction between two chemically separated tissue cultures. Human Micro Vascular Endothelial Cells (HMVECad) cultivated in vitro were used in experiments. The experiment was set after 24 hours of a sub-culturing the cells and the final setup was left for 48 hours in darkness of a CO2 incubator.
A Petri dish was placed on another one separated only by a polystyrene barrier. This setup ensures chemical separation but allows optical connection between the cultures. The culture in the upper dish was exposed to an oxidative stress caused by adding hydroxide peroxide. The stimulated culture acts as a source of photon emission which is presumed to carry information and to affect a cell division of a tissue culture in its neighborhood. Hence, the response of cells in the bottom dish kept under physiological conditions was studied. Medium with the same amount of hydroxide peroxide was used as a control sample in order to exclude effects of the reaction of medium with hydroxide peroxide itself. Evaluation was done by determination of the cell concentration using neutral red staining and employing a spectrophotometer.
In an early stage of the study we can supports a statement that the used tissue culture is capable of an interaction with another chemically separated culture via optical range of electromagnetic field.
Intercellular communication by light in unicellular alga Chlamydomonas reinhardtii
Parvez Ahmad and Pavel Pospíšil
Intercellular communication between cells is very important for the metabolic processes of the cells. Apart to communication by signal molecules as the most common type of cell-to-cell communication, evidence has been provided on cell-to-cell communication by light. In this study, the unicellular green alga Chlamydomonas reinhardtii have been used to study the communication between two types of cultures with different cell density: 1) high dense culture in the beginning of the stationary phase (3 days old) and 2) low dense culture at the beginning of the log phase (1 day old). The cell density was determined by direct cell counting by heamocytometer. In parallel to cell density, two-dimensional imaging of ultra-weak photon emission was measured using charge coupled device camera. Our results show that the cell density of high dense culture is increased, when high dense culture is in the optical contact with low dense culture and vice versa. The cell-to-cell communication correlates with two-dimensional imaging of ultra-weak photon emission. Based on these results, it is concluded that bio-communication between the high dense culture and low dense culture occurs in both directions.
Primo Vascular System and Optical Communication Channels of Ultraweak Photons
Almost fifty years ago, Bong-Han Kim first reported the existence of a third circulatory system in addition to the blood and lymphatic systems. He proposed that these novel anatomical vessels are distributed through out a body and those in the skin were acupuncture meridians. This system is now known as the primo vascular system (PVS). Since 2002 the long-neglected PVS has been reinvestigated in Seoul National University and there have been numerous descriptions of the primo vascular system which is comprised of primo-nodes (PN) and primo-vessels (PV). The PVS has been reported in various tissues in mice, rats and rabbits.
A hypothesis on long distance communication between organs through the acupuncture meridians and PVS was proposed with the optical channels of coherent ultraweak photons (UP) propagating in the primo vessels. The light sources are the primo micro-cells (or sanals) flowing in the primo vessels. Their medical functions are regeneration of cells like embryonic-like adult stem cells and contain DNA. These DNA are sources of UP in the PVS and can form active coherent light sources. Animals or humans can use these coherent UP for communication purpose of controlling the connected organs as a holistic system.
Biophotonic activities and transmission in neural circuits: methodological and theoretical considerations.
Jiapei Dai, Rendong Tang
The processing of neural information in neural circuits plays a key role in neural functions. It is well accepted that neuronal communication is mediated by bioelectricity and chemical molecules via the processes called bioelectrical and chemical transmission, respectively, which mainly occur in axons and synapses. Indeed, they seem to provide reasonable explanations for the basic functions and neural encoding of the nervous system, but many unanswered questions and debates remain. It has recently been suggested that biophotons, also called ultra-weak photon emissions, may play a potential role in neural signal transmission, contributing to the understanding of the high functions of nervous system; however, the experimental analysis of biophotonic activities (emissions) in neural circuits has been hampered due to technical limitations. By developing and optimizing an in vitro biophoton imaging method, we characterize the glutamate-induced biophotonic activities along the axons of cortical projection neurons and in the intrahippocampal circuits in mouse brain slices. We show that the long-lasting application of 50 mM glutamate to brain slices produces a gradual and significant increase of biophotonic activities and achieves the maximal effect within approximately 90 min, which then lasts for a relatively long time (> 200 min). The initiation and/or maintenance of biophotonic activities by glutamate can be obviously blocked by oxygen and glucose deprivation, together with the application of a cytochrome c oxidase inhibitor (sodium azide), but only partly by an action potential inhibitor (TTX), an anesthetic (procaine), or the removal of intracellular and extracellular Ca2+. The detected biophotonic activities mostly originate from axons, and the hyperphospholyation of microtubule-associated protein tau leads to a significant decrease of biophotonic activities in axons. Furthermore, the application of glutamate in the hippocampal dentate gyrus results in obvious biophotonic activities in its intrahippocampal projection areas. These results suggest that the glutamate-induced biophotonic activities reflect biophotonic transmission in neural circuits, which may be a new mechanism for the processing of neural information and neural functions. In addition, it seems that either bioelectrical or chemical transmission may only provide a basis for the initiation and maintenance of biophotonic activities and transmission.
Although our findings allow for the reconsideration of the traditional views on the roles of bioelectrical and chemical transmission, which are believed to be important mechanisms for the processing of neural information including the neural encoding and mechanisms of neuronal networks, however, some key questions remain to be answered. For example, what is the mechanism of biophotonic transmission in neural circuits? How are biophotons involved in synaptic signal transmission? How do we construct novel models for neural information coding according to biophotonic activities and transmission, and should quantum theory be considered based on photon behavior characteristics? The answers to these questions should help to explain the fundamental mechanisms of neural communications, and the functions of nervous system, such as vision, learning and memory, cognation and consciousness, and the mechanisms of human neurological and psychiatric diseases.
On the power of electromagnetic bio-communication
Throughout last 80 years researchers have claimed to observe electromagnetic signaling and communication between living cells. It this contribution we analyze the possibility of encoding information into properties of ultra-weak photon emission from cells. From comparison of reported experiments with information theory we conclude that phenomenon of electromagnetic bio-communication is very likely limited to very special circumstances.
Biophoton emissions from humans and cell cultures: A dynamic approach to biochemical processes in parallel systems.
Spontaneous biophoton emissions were measured from cellular and human subjects in complete darkness in multiple experimental conditions. These experiments have shown that a photomultiplier tube place 15 cm from the head can measure ultraweak photon energies in the range of 10-11 W/m2. Significant increases in ultraweak biophoton emissions from the right sides of volunteers’ heads were measured when they imagined white light in a dark environment compared to when they did not. Simultaneous variations in regional quantitative electroencephalographic spectral power (µV2/Hz) and total energy in the range of ~10-12 J from concurrent biophoton emissions were strongly correlated (r=0.95). The calculated energy was equivalent to that associated with action potentials from about 107 cerebral cortical neurons. In addition, metabolic activators like Genistein produced conspicuously high peaks in emission from cell cultures (B16-BL6) followed by an obvious drop in energy output. This pattern of emission suggests that the photon source was quantitatively limited and discrete. This would be characteristic of membrane potentials and potentially implicate the membrane as the primary source of emission. These results indicate that under specific conditions changes in photon emissions may reflect intercellular and interbrain communications with the potential for quantum-like properties.
Benefits of Ultraweak Photon Emission Technology for the Industry
Over the last twenty years the research on Ultraweak Photon Emission (UPE) has shown that a lot of information about the samples can be extracted from the measurements. The different research groups from all around the world have worked in different directions. From the measurements of brain and muscle activities, over the research on various cell cultures and skin samples, to the observations made on plants and seeds. A lot of data has been collected over the last years and the research is still going on. The food industry, the health care institutes and biotech industry have nowadays the possibility to use these results to have access to new quality control instruments and new diagnostic tools. The advantages of such a technology are obvious. The sort time of the measurements and the fact that the method stays non-invasive, are best examples for the benefits of the UPE technologies. But on the other side UPE brings also new challenges for the different industries. In terms of samples preparation and automation UPE measurements can become very demanding, especially when it comes to high rate measurements. The possibilities and challenges for the UPE technologies will be discussed during this presentation.
The Current State of the Art in Ultra-weak Photon Emission (UPE)
Roeland Van Wijk
Ultra-weak photon emission recordings from cells, tissues or entire living systems using photomultiplier tubes was initiated approximately 60 years ago. Its historic development is fascinating. It can be subdivided in three stages that can be recognized in the current state of the art.
1.) Initiation and globalization of UPE research of UPE research with photomultiplier tubes (PMT’s) and the radical oxygen connection. In 1960’s Russian groups headed by Tarusov, Vladimirov, Zhuravelv and Konev estimated UPE from all types of organisms. They continued to produce emission data in relationship to physiology and biochemistry. They demonstrated that photon emission is dependent on oxygen documenting their observations vis-à-vis chain oxidation reactions and reactive oxygen species. From the late 1960’s on, six major centers took birth in Japan, Poland, Australia, Germany, USA and China. It was the beginning of a worldwide research addressing UPE in living systems and the use of this emission as a sensitive assay for oxidative radical reactions.
2.) From internationalization to integration and the revival of the photon field concept. Research on electromagnetic fields and photon emission in living systems resulted from the late 1970’s on in a multidisciplinary collaboration. It resulted in the period 1977-1992 in several major symposia on UPE and in two multi-author reviews. These activities began to discern the richness of information which can be retrieved from measurements of photon emission. World-wide photon field research begins. Photon emission and storage in tumor cell line models demonstrated the link to the dualism growth – differentiation. It was the first link to the mitogenetic field as it was first proposed by Gurwitsch in response to the search for a field as part of early living system biology (in embryology, regeneration biology and tumor biology).
3.) Research on novel aspects in transmission and transduction of high energy endogenous to the living system began in the 1990’s. The research covered energy transfer at the level of the individual protein, DNA and the entire organism. It led to the expectation that UPE should be able to target the specific “optical window” of the electromagnetic interaction field and that detection of such emission would provide a unique “language” to facilitate more information about the biophysics of the living organization. Its applicability to health and disease began since the late 1990’s. Utilizing both PMT and 2-dimensional imaging systems accurate quantitative data regarding diseased states were captured. Human protocols were developed in order to record the body patterns of photon emission in relationship to health and disease as well as life style aspects. Other research developments connect animal and human photon emission with the levels of metabolomics and proteomics in diagnosis.
The current state in UPE research shows the products of developments in each of the three stages. The present research covers a broad spectrum of biological disciplines, from biochemistry (redox metabolism) to cell and developmental biology (photon field properties) to systems biology (in health and disease).
Ultra-weak photon emission as non-invasive system diagnostic tool: A proof of principle
Eduard P. Van Wijk, Herman A. van Wietmarschen, Roeland Van Wijk, Jan van der Greef,
The prevalence of type 2 diabetes continuously increases globally. It requires novel strategies to detect early stages in pre-diabetic typing that is vital for diabetic prevention and management. With that aim in mind, a large study is focusing on the combination of ultra-weak photon emission (intensity and distribution) and metabolomics (metabolite patterns in urine) in order to subtype pre-diabetic subjects and improve personalized treatment.
As a first step, an explorative study was carried out with 50 pre-diabetic males (fasting blood glucose ≥ 6.1 and ≤ 6.9 mmol/L; 30-70 years; BMI ³ 26 and £ 35 kg/m2). They were analyzed using ultra-weak photon emission (UPE) measurements and GC-MS urine metabolomics. Then, they were diagnosed by three physicians. The three physicians reached 85% diagnosis consistency resulting in the classification of 3 pre-diabetic subgroups. The subgroups were clearly distinct in their urine metabolic patterns. Although, no significant correlations between UPE and urine metabolites was observed in data of the entire group of 50 pre-diabetic males, there were significant correlation in the different subgroups. The present data will be used to study the power of the combination of UPE and metabolomics to discriminate different sub-types of pre-diabetes resulting in more personalized treatment.
The Application of Ultra Weak Photon Emission in Dermatology: UVA induced Oxidative Stress In Vivo
Oxidative stress and free radical reactions play a central role in skin aging and photo-aging. Ultra weak photon emission (UPE) serves as an effective in vivo tool in studying how does skin react to external oxidative stimuli and how does skin recover from the damage. We explored UVA induced skin oxidative stress by UPE and evaluated how does skin react to different UVA doses and fluence rates. We found that higher UVA dose in fact caused more oxidative stress in skin and it took longer to recover. In combination with other techniques, UPE may also help us address some of the key issues in skin photo-aging and provide valuable insight regarding the location and the source of the UVA induced oxidative stress in skin in vivo as well as the importance of oxygen accessibility. We proposed that collagen in dermis played a significant role in skin oxidative stress caused by UVA. These studies not only help us gain deeper understanding of the biological mechanisms such as skin pigmentation and photo-aging but also help us design better prevention and intervention protocols in areas such as sun protection and acne treatments.
Application of UPE in agriculture
Ultraweak photon emission (UPE) from living organisms including plants is well known to change its intensity responding to many kinds of stress. In plants, we have been developing techniques utilizing this character of UPE, to measure the state of plants non-invasively. Plant disease response against pathogen infection and plant response to herbicides were selected because of their importance in agriculture.
We first found that UPE increased based on disease response and from spectral analysis, it was speculated that this UPE derived from biochemical reactions quite different from those in the normal state of cells. To investigate further, we use rice cells vs. chitin elicitor system as a model for analyzing the relation between plant disease response and elicitor-responsive photon emissions (ERPE). Pharmacological analysis clarified that ERPE were generated through phosphatidic acid (PA), the second messenger leading to the reactive oxygen species (ROS) generation in the signal transduction of disease response. In addition, we found that the pattern of ERPE was almost identical to that of hydrogen peroxide generation, suggesting that ERPE are closely associated with the ROS-generating system.
Recently, plant activators, a new type of agricultural chemicals, are developed and used for disease protection. These plant activators can help plants to overcome pathogens by potentiating plant original disease response just after the recognition of pathogens. We successfully detect the activity of plant activators by observing the potentiation of EREP. Based on this principle, plant activator screening system was developed.
Currently, there are 393 biotypes in 211 weed species that have evolved resistance to compounds in all the major groups of herbicides. Recently we proposed a novel method for identifying herbicide resistance on the basis of UPE measurement.
Sulfonylurea (SU) herbicides are one of the most potent ALS-inhibiting herbicides used worldwide and biotypes resistant to SU herbicides have been found in many weed species. Mutations in the ALS genes, the target-site of SU herbicide, make weed species resistant to SU. This type of resistance is referred to as target-site resistance. However, there is another type of herbicide resistance, non-target-site resistance, which is caused by activation of herbicide metabolism, etc.. In both types of resistance, the resistant plants exhibited an increase in UPE after SU treatment. We found that oxidative metabolism by cytochrome P450 monooxygenase (P450) might be involved in UPE, based on the experiments of gene-silencing and inhibitors. It is considered that weeds resistant to herbicides other than SU might also be able to be distinguished from susceptible ones by UPE measurement, as long as the herbicides are subject to detoxification by P450 enzymes.
Ultra weak photon emission from Saccharomyces cerevisiae tubulin and mitochondrial mutants
Michal Cifra, Kateřina Červinková, Michaela Nerudová, Ondřej Kučera,
Daniel Havelka, Jiří Hašek
This works deals with measurement and analysis of ultra weak photon emission from Saccharomyces cerevisiae cold‐sensitive tubulin mutants tub2‐401 (strain CUY67 Mata tub2‐401 ura3‐52, ade2‐101) and ptc5Δ and pkp1Δ mitochondrial mutants. The exponentially growing cells of the tubulin mutant were placed into a glass vessel containing either in 2% sucrose or YPD (dextrose) medium. The mitochondrial mutant cells grown in YPD medium were transferred into either YPD or YPG (glycerol) medium. All cultures were incubated further in a light‐tight and temperature regulated chamber. Selected low noise photomultiplier tube R4220P housed in the C9144 cooler (both Hamamatsu Photonics K.K.) was mounted to the bottom of the light‐tight chamber as the photon detector. The dark count of the photomultiplier (operated at ‐25 OC) was around 0.07 counts per second. The culture of the tubulin mutant was splitt into two groups of cells. One of them was arrested (synchronized) before mitosis by incubation of the mutant cells at 14 OC for 4h. Another group of the mutant cells was cultivated at 30 OC and therefore the culture was not synchronized. Both groups were washed in sucrose and set to the same concentration before the measurement which took place at 28 OC. Photon emission from synchronized and non‐synchronized tubulin mutant cells was measured to check its dependence on the cell cycle synchronization. Mitochondrial mutants were measured either in YPD or YPG medium. The ptc5Δ mutant has deleted PTC5 gene which encodes phosphatase enzyme. Lacking phosphatase, pyruvate dehydrogenase is fully phosphorylated and inactive. Therefore, pyruvate is not decarboxylated to acetyl‐CoA. Since acetyl‐CoA is crucial for citric acid cycle, mitochondrial oxidative metabolism is significantly reduced. Reactive oxygen species, which lead to reactions generating photon emission, are known to be produced in significant amounts in mitochondria. Therefore, ptc5Δ mutants on glycerol are expected to produce lower photon emission than pkp1Δ mutants under same conditions. Statistical methods were used to characterize the results from all measurements.
Statistics of biophoton emission from developing eggs and embryos
Ilya V. Volodyaev Lev V. Beloussov.
Biophoton emission (BPE) from Xenopus eggs and embryos was measured with photoelectromultiplier, using signal accumulation time from 1 ms to 1 s. We show that BPE consists of single flashes of no more than millisecond duration, which altogether occupy less than 1% of total measurement time, producing from 0.6 to 7 flashes per second. Among eggs and early embryos, the greatest BPE intensity (both by total photon number and average value of single flashes) was adjusted to the period of egg fertilization, first cleavage divisions, mid-late blastula and gastrulation. The burst of BPE intensity at blastula stage coincides with the time of activation of zygotic genes. A biological role of highly intermittent BPE regime is discussed.
Information Encoding in bio-photon communications; ORLANDO project
Encoding algorithm of bio-photon signals was explored using the data on the optical radiation from fish eggs (Misgurnusfossilis) which were obtained previously by BMN collaboration . It was found that such radiation is emitted in form of short ( about 10-3 sec or less) quasi-periodic bursts with the typical intervals .1 ÷ 10 sec between them . Hence its temporary structure is similar to the binary encoded messages in which each such burst transfers one bit of information. The sample of about 200 fish eggs was confined in quartz container filled by water; the optical radiation up to 200 nm from container was detected by the photomultiplier. Its integral photocurrent over consequent .1 sec intervals was recorded. Fourier analysis of photocurrent demonstrate that the radiation from fish eggs is essentially more structurized and periodical in comparison with background radiation, in particular, the average value of fourier autocorrelation over 102 sec is 40% larger. It was found also that for burst amplitudes higher than 20 arbitrary units, the average time period T between them decreases proportionally to egg age varied from 15 up to 40 hours from 10.6 ± .6 sec to 6.2 ± .4 sec. Thus, the observed T difference encodes the information about the development stage. For fish eggs such signals supposedly synchronize the development between distant parts of egg colony, the existence of such effect was confirmed experimentally .
ORLANDO project continues the investigation of bio-photon communications and their possible applications in medical diagnostics and other bio-technology problems. In particular, it's planned to compare bio-communications effects in lab. conditions and on the earth orbit. For that purpose the specialized module will be delivered and installed on International Space Station. In this set-up array of silicon photodiods detects the radiation of several different biological samples - inductors. Simultaneously, mitogenetic effect will be investigated performing inductor optical contact with other biological sample - detector and measuring the rate of cell division in it.
 - Moraes, Thiago A. ; Barlow, Peter W ; Klingelé, Emile ; Gallep, Cristiano M. . Spontaneous ultra-weak light emissions from wheat seedlings are rhythmic and synchronized with the time profile of the local gravimetric tide. Naturwissenschaften, v. 99, p. 465-472, 2012. http://dx.doi.org/10.1007/s00114-012-0921-5.
 - GALLEP, Cristiano de Mello ; Moraes, Thiago A. ; SANTOS, Samuel Ricardo dos ; Barlow, Peter W . Coincidence of biophoton emission by wheat seedlings during simultaneous, transcontinental germination tests. Protoplasma, p. s00709-012-0447, 2012.
 - GALLEP, Cristiano de Mello ; SANTOS, Samuel Ricardo dos . Photon-counts during germination of wheat (Triticum aestivum) in wastewater sediment solutions correlated with seedling growth. Seed Science and Technology, v. 35, p. 607-614, 2007.
 - BERTOGNA, E. ; BEZERRA, J. ; CONFORTI, E. ; GALLEP, C.M. . Acute stress in seedlings detected by ultra-weak photon emission. Journal of Photochemistry and Photobiology. B, Biology, v. 118, p. 74-76, 2013. http://dx.doi.org/10.1016/j.jphotobiol.2012.11.005
 - BERTONHA, E. ; SANTOS, Samuel Ricardo dos ; CONFORTI, Evandro ; GALLEP, Cristiano de Mello . Compact, Automatic Set-Up for Ultra-Weak Photon Emission Measurements in Organisms . In: 2011 SBMO/IEEE MTT-S IMOC 2011, 2011, Natal. proc. of, 2011. v. 1.