Super-high sensitivity systems for detection and spectral analysis of ultraweak photon emission from biological cell cells and tissues

Summary In this paper we summarize and discuss the modern technology and systems, studied and established by our research group, for performing the detection and special analysis incorporated with the super-high senstivity photon counting method for the study of ultraweak photon emission; for example, extra-weak bioluminescence and chemiluminescence from living cells and tissues, closely related to biochemical and biophysical processes and activities. An excellent sensitivity of the basic photon counting system, making it possible to achieve count rates in the very low range of one photoelectron per second to one per minute, allowed us to carry out in vivo as well as in vitro measurements, and analyses of ultraweak bioluminescence and chemiluminescence. Recent results concerning ultraweak photon emission from blood samples and organ homogenates of rats are presented and reviewed as one of the interesting and valuable applications of our modern technology for studying ultraweak cell and tissue radiation.     

Experimental study on photocount statistics of the ultraweak photon emission from some living organisms

The hypothesis that biophotons display a high degree of coherence was tested by measuring photocount statistics (PCS) of the ultraweak photon emission from three living organisms (cucumber seedling, mungbean seedling and soybean rhizobium bacteroids) with a high-sensitivity single-photon counter. For comparison, the same experiments were performed for laser beam, randomized laser beam, chemiluminescence from autoxidation of luminol and the dark counts of the equipment. Photocount distributions, close to Poissonian, were observed for the three tested biological systems but not for the pure chemiluminescence of luminol.     

Regulatory aspects of low intensity photon emission

Photon emission from unicellular and multicellular organisms has been a subject of study for many decennia. In contrast to the well-known phenomenon of bioluminescence originating in luciferin-luciferase reactions, low intensity emission in the visible region of the electromagnetic spectrum has been found in almost every species studied so far. At present, the nomenclature of this phenomenon has not crystallized and it is referred to by a variety of names, such as mitogenetic radiation 29, dark luminescence 7, low-level chemiluminescence 20,36, and biophotons 57. Particular attention has been focussed on the relationship between photon emission and the regulation of various aspects of cellular metabolism, although in many cases quantitative data are still lacking. Throughout the history of this field of research the question of a functional biological role of the low intensity emission has been repeatedly raised; this is reflected, for instance, in the heterogeneity of the terms used to describe it. The discussion concerns the possible participation of photons of low intensity in intra- and intercellular communication. This paper reviews literature on the metabolic regulation of low intensity emission, as well as the regulation of photon emission initiated by external light. Furthermore, recent data are discussed with respect to a possible biocommunicative function of low intensity photon emission.     

The effect of stress factors on the spontaneous photon emission from microorganisms

The results of recent work on the photon emission from three yeasts and a bacterium is presented. Both visible region and ultraviolet photon emission is observed; however, no luminescence is observed in the absence of oxygen. The visible region emission is attributed to excited carbonyl groups and excited singlet oxygen dimers formed during the decomposition of lipid hydroperoxides. Possible sources of the ultraviolet photon emission are also examined. The use of microorganisms in the study of ultraweak photon emission and its relation to oxidative, temperature and chemical stress is reviewed and the applications and (or) functions of this photon emission are also discussed.     

The effect of stress factors on the spontaneous photon emission from microorganisms.

The results of recent work on the photon emission from three yeasts and a bacterium is presented. Both visible region and ultraviolet photon emission is observed; however, no luminescence is observed in the absence of oxygen. The visible region emission is attributed to excited carbonyl groups and excited singlet oxygen dimers formed during the decomposition of lipid hydroperoxides. Possible sources of the ultraviolet photon emission are also examined. The use of microorganisms in the study of ultraweak photon emission and its relation to oxidative, temperature and chemical stress is reviewed and the applications and (or) functions of this photon emission are also discussed.     

Photon emission in tumor biology

Photon emission from mammalian cells has been subject of study for many years. Growing research activity is directed on the photon emission within the field of tumor biology. These studies, applying high-sensitivity photon counting methods, have paid attention to several aspects, including photon emission from serum of tumor-bearing animals, photon emission of tumors and of isolated tumor cells. In addition, research activity is increased with respect to the photon emission by white light from cultured tumor cells. In this review we report on the different aspects of spontaneous and induced photon emission of tumor cells as compared to normal cells. Throughout these studies the question of a functional biological role of this spontaneous and light-induced photon emission has been raised and some different points of view will be discussed.     

Photon emission in tumor biology.

Photon emission from mammalian cells has been subject of study for many years. Growing research activity is directed on the photon emission within the field of tumor biology. These studies, applying high-sensitivity photon counting methods, have paid attention to several aspects, including photon emission from serum of tumor-bearing animals, photon emission of tumors and of isolated tumor cells. In addition, research activity is increased with respect to the photon emission induced by white light from cultured tumor cells. In this review we report on the different aspects of spontaneous and induced photon emission of tumor cells as compared to normal cells. Throughout these studies the question of a functional biological role of this spontaneous and light-induced photon emission has been raised and some different points of view will be discussed.     

Stress-induced photon emission from perturbed organisms

This paper reviews an ultraweak luminescent response of selected biological systems (lower and higher plants, insects and spermatozoa) to certain kinds of detrimental mechanical, temperature, chemical and photochemical stress and to lethal factors. The enhancing effect of white light and formaldehyde on the ultraweak luminescence of yeast and spermatozoa cells is described for the first time. An increase in the percentage of long wavelengths (λ>600 nm) with an increase in reaction time, and a significant influence of the suspending medium on the ultraweak luminescence, were observed. The vitality and motility of bull spermatozoa and the vitality of yeast cells were drastically decreased by treatment with white light, water, formaldehyde and iron-ions. Successive irradiation of intact bull spermatozoa cells with white light caused an increase in the intensity of delayed luminescence. An attempt has been undertaken to find stochastic models of non-stationary photon emission. The quasi-relaxation descending stage of non-stationary processes can be modeled as the Integrated Moving Average process IMA (0,1,1), and memory and transfer functions can describe the degree of perturbation in the yeastSaccharomyces cerevisiae. The relation of the ultraweak luminescence response to perturbations of homeostasis is discussed in the framework of biochemical and physical models.     

Stress-induced photon emission from perturbed organisms.

This paper reviews an ultraweak luminescent response of selected biological systems (lower and higher plants, insects and spermatozoa) to certain kinds of detrimental mechanical, temperature, chemical and photochemical stress and to lethal factors. The enhancing effect of white light and formaldehyde on the ultraweak luminescence of yeast and spermatozoa cells is described for the first time. An increase in the percentage of long wavelengths (lambda > 600 nm) with an increase in reaction time, and a significant influence of the suspending medium on the ultraweak luminescence, were observed. The vitality and motility of bull spermatozoa and the vitality of yeast cells were drastically decreased by treatment with white light, water, formaldehyde and iron-ions. Successive irradiation of intact bull spermatozoa cells with white light caused an increase in the intensity of delayed luminescence. An attempt has been undertaken to find stochastic models of non-stationary photon emission. The quasi-relaxation descending stage of non-stationary processes can be modeled as the Integrated Moving Average process IMA (0, 1, 1), and memory and transfer functions can describe the degree of perturbation in the yeast Saccharomyces cerevisiae. The relation of the ultraweak luminescence response to perturbations of homeostasis is discussed in the framework of biochemical and physical models.     

Biophoton emission fromDaphnia magna: A possible factor in the self-regulation of swarming

Summary The formation of swarms by planktonic organisms was first described almost 100 years ago, but the mechanisms governing the development of patterns in population size and density are still not understood. In this study, we investigated one biophysical factor that may play an important role in swarm-formation. Spontaneous ultraweak photon emission in the visible range has been well documented for living cells, tissues and individuals in the plant and animal kingdom, including humans. We demonstrate here that the intensity of light emitted by the planktonic crustaceanDaphnia magna is a function of population density in relation to body size. The effects are discussed on the basis of the theory of Dicke^1,2, and it is suggested that biophoton emission may be a basic factor in the self-regulation of swarm density.     

Regulatory aspects of low intensity photon emission

Summary Photon emission from unicellular and multicellular organisms has been a subject of study for many decennia. In contrast to the well-known phenomenon of bioluminescence originating in luciferin-luciferase reactions, low intensity emission in the visible region of the electromagnetic spectrum has been found in almost every species studied so far. At present, the nomenclature of this phenomenon has not crystallized and it is referred to by a variety of names, such as mitogenetic radiation²⁹, dark luminescence⁷, low-level chemiluminescence^{20, 36}, and biophotons⁵⁷. Particular attention has been focussed on the relationship between photon emission and the regulation of various aspects of cellular metabolism, although in many cases quantitative data are still lacking. Throughout the history of this field of research the question of a functional biological role of the low intensity emission has been repeatedly raised; this is reflected, for instance, in the heterogeneity of the terms used to describe it. The discussion concerns the possible participation of photons of low intensity in intra- and intercellular communication. This paper reviews literature on the metabolic regulation of low intensity emission, as well as the regulation of photon emission initiated by external light. Furthermore, recent data are discussed with respect to a possible biocommunicative function of low intensity photon emission.     

The origin,diversity, and structure function relationships of insect luciferases

Luciferases are the enzymes that catalyze the reactions that produce light in bioluminescence. Whereas the oxidative mechanism which leads to light emission is similar for most luciferases, these enzymes and their substrates are evolutionarily unrelated. Among all bioluminescent groups, insects constitute one of the most diverse in terms of biochemistry. In the fungus-gnats (Mycetophilidae: Diptera), for example, bioluminescence is generated by two biochemically distinct systems. Despite the diversity, investigations on insect luciferases and biochemistry have been conducted mostly with fireflies. The luciferases from the related phengodid beetles, which can produce green to red bioluminescence using the same chemistry as firefly luciferases, have been recently investigated. Beetle luciferases originated from ancestral acyl-CoA ligases. Present data suggest that conserved motifs among this class of ligases are involved in substrate adenylation. The three-dimensional structure of firefly luciferase was recently solved and mutagenesis studies have been performed identifying putative residues involved in luciferin binding and bioluminescence color determination in several beetle luciferases. The knowledge gained through these studies is helping in the development of useful reporter gene tools for biotechnological and biomedical purposes. Received 4 March 2002; received after revision 13 May 2002; accepted 21 May 2002     

生物活性物质的电致化学发光检测

电致化学发光是通过电极上直接或间接发生的电化学反应而产生的一种化学发光,因此电致化学发光检测是在化学发光和电化学基础上发展起来的一种新的分析技术。电致化学发光检测技术不但保留了化学发光分析和电化学分析固有的优点,同时还具有其自身的优点,如所发生的化学发光反应易于控制;方法更灵敏,更具有选择性;可以获得更多的化学信息;扩大了化学发光方法可检测的范围;更易于与现代分离技术联用。生物体中很多生物活性物质具有电活性,因此用现代电化学技术研究其电化学行为具有重要的理论意义和实际应用价值。生物体中的生物活性物质通常浓度很低,并且成分复杂,因此分离检测生物体中生物活性物质非常困难。由于电致化学发光检测具有灵敏度高,选择性好的特点,无疑是检测生物体中生物活性物质的强有力工具,如果它能与HPLC、CE及FIA等现代分离技术相结合,将表现出更加强大的生命力。     

Molecular enigma of multicolor bioluminescence of firefly luciferase

Firefly luciferase-catalyzed reaction proceeds via the initial formation of an enzyme-bound luciferyl adenylate intermediate. The chemical origin of the color modulation in firefly bioluminescence has not been understood until recently. The presence of the same luciferin molecule, in combination with various mutated forms of luciferase, can emit light at slightly different wavelengths, ranging from red to yellow to green. A historical perspective of development in understanding of color emission mechanism is presented. To explain the variation in the color of the bioluminescence, different factors have been discussed and five hypotheses proposed for firefly bioluminescence color. On the basis of recent results, light-color modulation mechanism of firefly luciferase propose that the light emitter is the excited singlet state of OL⁻ [¹(OL⁻)*], and light emission from ¹(OL⁻)* is modulated by the polarity of the active-site environment at the phenol/phenolate terminal of the benzothiazole fragment in oxyluciferin.     

UV guided dendritic growth patterns and the networking of melanocytes

Whole skin organ cultures of vitiliginous, skin show that the marginal melanocytes are highly sensitive to a pulse of UV exposure (210–380 nm) during the G₂ phase of the cell cycle, as seen by prominent dendricity. Melanocytes are highly dendritic in the epidermis overlying rapidly growing tumors, as well as within proliferative lesions such as basal cell carcinomas and aggressive seborrheic keratosis. In the organ cultures the dendrites extend towards the source of UV, i.e. the surface, while the main body lies along the basement membrane. The epidermal melanocytes overlying tumors lie, almost vertically, dendrites aligned towards the underlying tumor on one side and the surface on the other. Within tumors dendritic elongation is guided by mitotic and PCNA positive (S-phase) tumor cells, which are a source of ultraweak UV emissions in the range of 210–330 nm. These observations indicate that ultraweak biophoton emissions from neighbouring cells can simulate environmental cues and contribute to the plasticity of networks such as the melanocytes or the visual pathways.     

过氧草酰类化学发光分析研究的新进展

过氧草酰类化学发光体系具有很高的发光效率并且适于多种类型物质检测。本文对此类发光试剂在分析化学领域的最新研究进行评述，主要内容包括发光机理的研究，新试剂的合成以及在分析检测中的应用。     

Luminescence research and its relation to ultraweak cell radiation

The fundamental laws of photochemistry and the essential results of experimental research on ultraweak cell radiation are presented. By comparing all the facts it can be concluded that the phenomena discussed may arise from a variety of possible reactions and sources. Recombination reactions of certain radicals actually do release sufficient energy to generate UV-photons of the intensity under consideration. On the other hand, stimulated emission cannot be excluded in view of the distinct deviation of the radiation field from thermal equilibrium. There exist, however, various other candidates, such as direct emitters like flavins, indoles, porphyrins, carbonyl derivatives and aromatic compounds, and molecular oxygen and its various species, as well as collective molecular interactions, e.g. dimole or exciplex transitions, triplet-triplet annihilation, collective hydrolysis, electric field effects in membranes, etc. Careful biochemical and biophysical experiments are still necessary to find answers to all the questions that remain; not only individual problems have to be solved, but it is important to keep in mind the interrelationships between certain reactions.     

Inhibition of firefly bioluminescence by scavengers of singlet oxygen,superoxide radicals and hydroxyl radicals

Summary The participation of highly energetic oxygen species in the ATP-induced bioluminescence of a firefly-extract has been investigated. The inhibition of light emission by a variety of specific scavengers suggests that singlet oxygen, superoxide radicals and hydroxyl radicals are important intermediates in the firefly bioluminescence reaction.Acknowledgments. I thank Prof. R. Bachofen, Institut für Allgemeine Botanik, Abteilung Mikrobiologie, Universität Zürich, Switzerland, in whose laboratory most of these studies have been performed, for his cooperativity. Financial support by the Deutsche Studienstiftung is gratefully acknowledged.     

Luminescence research and its relation to ultraweak cell radiation

Summary The fundamental laws of photochemistry and the essential results of experimental research on ultraweak cell radiation are presented. By comparing all the facts it can be concluded that the phenomena discussed may arise from a variety of possible reactions and sources. Recombination reactions of certain radicals actually do release sufficient energy to generate UV-photons of the intensity under consideration. On the other hand, stimulated emission cannot be excluded in view of the distinct deviation of the radiation field from thermal equilibrium. There exist, however, various other candidates, such as direct emitters like flavins, indoles, porphyrins, carbonyl derivatives and aromatic compounds, and molecular oxygen and its various species, as well as collective molecular interactions, e.g. dimole or exciplex transitions, triplet-triplet annihilation, collective hydrolysis, electric field effects in membranes, etc.Careful biochemical and biophysical experiments are still necessary to find answers to all the questions that remain; not only individual problems have to be solved, but it is important to keep in mind the interrelationships between certain reactions.