Theory of light quenching: effects of fluorescence polarization, intensity, and anisotropy decays.

Experimental studies have recently demonstrated that fluorescence emission can be quenched by laser light pulses from modern high repetition rate lasers, a phenomenon we call "light quenching." We now describe the theory of light quenching and some of its effects on the steady-state and time-resolved intensity and anisotropy decays of fluorophores. Light quenching can decrease or increase the steady-state or time-zero anisotropy. Remarkably, the light quenching can break the usual z axis symmetry of the excited-state population, and the emission polarization can range from -1 to +1 under selected conditions. The measured anisotropy (or polarization) depends upon whether the observation axis is parallel or perpendicular to the propagation direction of the light quenching beam. The effects of light quenching are different for a single pulse, which results in both excitation and quenching, as compared with a time-delayed quenching pulse. Time-delayed light quenching pulses can result in step-like changes in the time-dependent intensity or anisotropy and are predicted to cause oscillations in the frequency-domain intensity and anisotropy decays. The increasing availability of pulsed laser sources offers the opportunity for a new class of two-pulse or multiple-pulse experiments where the sample is prepared by an excitation pulse, the excited state population is modified by the quenching pulse(s), followed by time- or frequency-domain measurements of the resulting emission.     

Modification of Spontaneous Emission Rates of Self-assembled CdSe Quantum Dots by Coupling to Hybrid Optical Nanoantennas

Plasmonics - The spontaneous emission of a light source can be modified by tailoring its local density of optical states. Indeed, this concept has been commonly utilized to enhance the spontaneous...     

A new type of thermoluminometer: a highly sensitive tool in applied photosynthesis research and plant stress physiology

Here we describe a newly developed thermoluminescence measuring device that employs flash excitation, peltier heating, and light detection by channel photomultipliers (CPM). The new thermoluminometer is equipped with four sample holders for simultaneous measurements of thermoinduced light emission in the temperature range from -20 degrees C to +180 degrees C. It allows one to measure leaf samples, chloroplasts, thylakoids, algae, or even bioorganic material lacking chlorophyll by means of naturally induced or artificially applied chemilumigenic probes. The temperature range of the thermoluminometer allows one to analyse the thermoinduced radical pair recombination of photosystem II in the lower temperature region as well as chemiluminescence from lipid peroxidation in the higher temperature region. Hence, plant material can be assessed concerning both its photosynthetic and its oxidative stress status. Since the device is equipped with four sample holders and four CPM channels for simultaneous detection of thermoinduced light emission, it facilitates a high throughput. Therefore, the new device is interesting, not only in ecophysiology, but also in the field of plant breeding, as it can be used to study the stress tolerance of various cultivars of cultural crop plants.     

Biophoton emission. New evidence for coherence and DNA as source

The phenomenon of ultraweak photon emission from living systems was further investigated in order to elucidate the physical properties of this radiation and its possible source. We obtained evidence that the light has a high degree of coherence because of (1) its photon count statistics, (2) its spectral distribution, (3) its decay behavior after exposure to light illumination, and (4) its transparency through optically thick materials. Moreover, DNA is apparently at least an important source, since conformational changes induced with ethidium bromide in vivo are clearly reflected by changes of the photon emission of cells. The physical properties of the radiation are described, taking DNA as an exciplex laser system, where a stable state can be reached far from thermal equilibrium at threshold.     

Biophoton emission

The phenomenon of ultraweak photon emission from living systems was further investigated in order to elucidate the physical properties of this radiation and its possible source. We obtained evidence that the light has a high degree of coherence because of (1) its photon count statistics, (2) its spectral distribution, (3) its decay behavior after exposure to light illumination, and (4) its transparency through optically thick materials. Moroever, DNA is apparently at least an important source, since conformational changes induced with ethidium bromide in vivo are clearly reflected by changes of the photon emission of cells. The physical properties of the radiation are described, taking DNA as an exciplex laser system, where a stable state can be reached far from thermal equilibrium at threshold.     

The effect of luciferase and NADH:FMN oxidoreductase concentrations on the light kinetics of bacterial bioluminescence

The effects of NADH:FMN oxidoreductase and luciferase concentrations on the light kinetics of the bacterial bioluminescent reaction were investigated. Light emission with low decay rates was obtained by regulating the conversion of NADH to NAD+ by controlling oxidoreductase activity. Constant light emission can be obtained when the oxidoreductase activity is below 2.5 U/1 in the assay system. The luciferase concentration affects the light intensity but it has no effect on the decay rate of light emission. The substrate decanal and the end-products NAD+ and capric acid had no effect on the light kinetics. The Michaelis constants of bacterial luciferase for FMNH2 and decanal were 3 X 10(-6) M and 8 X 10(-7) M, respectively, and those of oxidoreductase for FMN and NADH were 6.1 X 10(-6) M and 1.6 X 10(-5) M, respectively.     

Bioluminescence characteristics of the fruiting body of Mycena chlorophos

Bioluminescent fungi are widely distributed on land and most belong to the class Basidomycetes. Light of about 530 nm wavelength maximum is emitted continuously. The molecular basis for the light‐emitting process remains unclear. We investigated the characteristics of the bioluminescence using cultivated fruiting bodies of M. chlorophos. Only fresh fruiting bodies exhibited long‐lasting light emission; rapid decay of light emission was observed with frozen and freeze‐dried samples. Freeze‐dried samples can be stored at room temperature under dry conditions and may be useful for the isolation of luciferin. The light emission of the fresh fruiting bodies was maintained in various buffers at varying pH; it could be stopped with pH 4 acetate buffer and could be recovered at pH 6. The isolation of luciferin from the fresh fruiting bodies might be possible by the control of buffer pH. The effect of temperature on the light emission of fruiting bodies indicated that bioluminescence in M. chlorophos may involve enzymatic reaction(s). The solubilization of bioluminescent components from the fruiting bodies could not be achieved with various surfactants. Copyright © 2011 John Wiley & Sons, Ltd.     

Process-based modelling of isoprene emission by oak leaves

The emission rate of the volatile reactive compound isoprene, emitted predominantly by trees, must be known before the level of photo‐oxidants produced during summer smog can be predicted reliably. The emission is dependent on plant species and local conditions, and these dependencies must be quantified to be included in any empirical algorithm for the calculation of isoprene production. Experimental measurements of isoprene emission rates are expensive, however, and existing data are scarce and fragmentary. To overcome these difficulties, it is promising to develop a numerical model capable of precisely calculating the isoprene emission by trees for diverse ecosystems, even under changing environmental conditions. A basic process‐based biochemical isoprene emission model (BIM) has therefore been developed, which describes the enzymatic reactions in leaf chloroplasts leading to the formation of isoprene under varying environmental conditions (e.g. light intensity, temperature). Concentrations of the precursors of isoprene formation, 3‐phosphoglyceric acid and glyceraldehyde 3‐phosphate, are provided by a published light fleck photosynthesis model. Specific leaf and enzyme parameters were determined for the pedunculate oak (Quercus robur L.), so that the BIM is capable of calculating oak‐specific isoprene emission rates as influenced by the leaf temperature and light intensity. High correlation was observed between isoprene emission rates calculated by the BIM and the diurnal isoprene emission rates of leaves measured under controlled environmental conditions. The BIM was even capable of describing changes in isoprene emission caused by midday depression of net photosynthesis.     

Induction of a longer term component of isoprene release in darkened aspen leaves: origin and regulation under different environmental conditions

After darkening, isoprene emission continues for 20 to 30 min following biphasic kinetics. The initial dark release of isoprene (postillumination emission), for 200 to 300 s, occurs mainly at the expense of its immediate substrate, dimethylallyldiphosphate (DMADP), but the origin and controls of the secondary burst of isoprene release (dark-induced emission) between approximately 300 and 1,500 s, are not entirely understood. We used a fast-response gas-exchange system to characterize the controls of dark-induced isoprene emission by light, temperature, and CO(2) and oxygen concentrations preceding leaf darkening and the effects of short light pulses and changing gas concentrations during dark-induced isoprene release in hybrid aspen (Populus tremula × Populus tremuloides). The effect of the 2-C-methyl-D-erythritol-4-phosphate pathway inhibitor fosmidomycin was also investigated. The integral of postillumination isoprene release was considered to constitute the DMADP pool size, while the integral of dark-induced emission was defined as the "dark" pool. Overall, the steady-state emission rate in light and the maximum dark-induced emission rate responded similarly to variations in preceding environmental drivers and atmospheric composition, increasing with increasing light, having maxima at approximately 40 °C and close to the CO(2) compensation point, and were suppressed by lack of oxygen. The DMADP and dark pool sizes were also similar through their environmental dependencies, except for high temperatures, where the dark pool significantly exceeded the DMADP pool. Isoprene release could be enhanced by short lightflecks early during dark-induced isoprene release, but not at later stages. Fosmidomycin strongly suppressed both the isoprene emission rates in light and in the dark, but the dark pool was only moderately affected. These results demonstrate a strong correspondence between the steady-state isoprene emission in light and the dark-induced emission and suggest that the dark pool reflects the total pool size of 2-C-methyl-d-erythritol-4-phosphate pathway metabolites upstream of DMADP. These metabolites are converted to isoprene as soon as ATP and NADPH become available, likely by dark activation of chloroplastic glycolysis and chlororespiration.     

Chemiluminescent determination of acetylcholine,and continuous detection of its release from torpedo electric organ synapses and synaptosomes

A chemiluminescent procedure to determine acetylcholine is described. The enzyme choline oxidase recently purified, oxidises choline to betaine, the H₂O₂ generated is continuously measured with the luminol-peroxidase chemiluminescent reaction for H₂O₂. Other chemi or bioluminescent detectors for H₂O₂ would probably work as well. The chemiluminescent step provides great sensitivity to the method which is slightly less sensitive than the leech bio-assay but much more sensitive than the frog rectus preparation. The specificity of the chemiluminescent method depends on the fact that choline oxidase receives its substrate only when acetylcholine is hydrolysed by acetylcholinesterase. The acetylcholine content of tissue extracts was determined with the chemiluminescent method, and with the frog rectus assay, the values found were very comparable. The chemiluminescent procedure was used to follow the release of acetylcholine from tissues. When a slice of electric organ is incubated with choline oxidase, luminol and peroxidase, KCl depolarization or electrical stimulation in critical experimental conditions triggered an important light emission, which was blocked in high Mg²⁺. The venom of Glycera convoluta, known to induce a substantial transmitter release, was also found to trigger the light emission from tissue slices. Suspensions of synaptosomes release relatively large amounts of acetylcholine following Glycera venom action; this was confirmed with the chemiluminescent reaction. The demonstration that the light emission reflects the release of acetylcholine is supported by several observations. First, when the tissue is omitted no light emission is triggered after KCl or venom addition to the reagents. Second, the time course of the light emission record is very similar to the time course previously found for ACh release with radioactive methods. Third, if choline oxidase is omitted, or if acetylcholinesterase is inhibited by phospholine, the light emission is blocked, showing that the substance released has to be hydrolyzed by acetylcholinesterase and oxidised by choline oxidase to generate chemiluminescence.The procedure described has important potential applications since other transmitters can similarly be measured upon changing the oxidase.     

Visible chemiluminescence from rat brain homogenates undergoing autoxidation. I. Effect of additives and products accumulation

Rat brain homogenate autoxidation was assessed from thiobarbituric acid reactant accumulation (TBAR), light emission, and oxygen uptake, The effect of several additives upon TBAR accumulation and light intensity suggests that these parameters can be employed as a reliable measure of the lipoperoxidation extent. From the different time profiles of TBAR accumulation and light emission, it is concluded that instantaneous light emission is not a measure of the lipoperoxidation rate but it is related to the accumulation of products. The time dependence of the light emitted after addition to an incubated sample of an excess of free radical scavengers indicates that at least two intermediates of widely different lifetimes are contributing to the observed light emission.     

Diurnal regulation of scent emission in rose flowers

Previous studies have shown diurnal oscillation of scent emission in rose flowers with a peak during the day (Helsper in Planta 207:88–95, 1998; Picone in Planta 219:468–478, 2004). Here, we studied the regulation of scent production and emission in Rosa hybrida cv. Fragrant Cloud during the daily cycle and focused on two terpenoid compounds, germacrene D and geranyl acetate, whose biosynthetic genes have been characterized by us previously. The emission of geranyl acetate oscillated during the daily light/dark cycle with a peak early in the light period. A similar daily fluctuation was found in the endogenous level of this compound and in the expression of its biosynthetic gene, alcohol acetyl transferase (RhAAT). The rhythmic expression of RhAAT continued under conditions of constant light or darkness, indicating regulation by the endogenous circadian clock. However, the accumulation and emission of geranyl acetate ceased under continuous light. Our results suggest that geranyl acetate production is limited by the level of its substrate geraniol, which is suppressed under constant light conditions. The emission of germacrene D also oscillated during the daily cycle with a peak early in the light period. However, the endogenous level of this compound and the expression of its biosynthetic gene germacrene D synthase (RhGDS) were constant throughout the day. The diurnal oscillation of germacrene D emission ceased under continuous light, suggesting direct regulation by light. Our results demonstrate the complexity of the diurnal regulation of scent emission: although the daily emission of most scent compounds is synchronized, various independently evolved mechanisms control the production, accumulation and release of different volatiles. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Guanosine triphosphate analysis: PEI-thin layer purification and luciferin-luciferase liquid scintillation quantitation

A novel method to purify and quantitate GTP is described. This method is simple to run and sensitive to detect GTP in picomole quantities. It is based on purfication and isolation of GTP on PEI-thin layer chromatograms and quantitation with the firefly luciferin-luciferase system.GTP characteristically gives a delayed (about 2 min) peak light emission similar to adenosine tetraphosphate (about 6 min) but different from ATP which gives a peak light emission value at zero time. The light emission from each of these three nucleotides is characteristically different from one another such that contamination between these three nucleotides can be detected by their peak light emission times. This method has been applied to analyze GTP in biological preparations which appeared to be free of detectable concentration(s) of interfering substances(s).     

Regulatory mutants of the Vibrio harveyi lux system

Regulatory mutants of the luminescent bacterium, Vibrio harveyi, have been isolated whose light emission can be stimulated by extracts of the growth media. Chloroform extracts of conditioned media in which V. harveyi has been grown can increase light emission in one of the dark mutants, D34, over 10³-fold. An increase in the level of the mRNA and the enzymes associated with the lux system can also be demonstrated. Analysis of the expression of the lux system in Escherichia coli transformed with DNA from the D34 regulatory mutant demonstrates that the mutation resides outside the luciferase structural genes. The results suggest that the decrease in light emission in the regulatory mutants may be due to a mutation in synthesis of an autoinducer analogous to that found for the Vibrio fischeri lux system.     

Enhanced luciferin entry causes rapid wound-induced light emission in plants expressing high levels of luciferase

In-vivo imaging of transgenic tobacco plants (Nicotiana tobacum L.) expressing firefly luciferase under the control of the Arabidopsis phenylalanine ammonia-lyase 1 (PAL1)-promoter showed that luciferase-catalyzed light emission began immediately after the substrate luciferin was sprayed onto the leaves and reached a plateau phase after approximately 60 min. This luminescence could easily be detected for up to 24 h after luciferin application although the light intensity declined continuously during this period. A strong and rapid increase in light emission was observed within the first minutes after wounding of luciferin-sprayed leaves. However, these data did not correlate with luciferase activity analysed by an in-vitro enzyme assay. In addition, Arabidopsis plants expressing luciferase under the control of the constitutive 35S-promoter showed similar wound-induced light emission. In experiments in which only parts of the leaves were sprayed with luciferin solutions, it was shown that increased uptake of luciferin at the wound site and its transport through vascular tissue were the main reasons for the rapid burst of light produced by preformed luciferase activity. These data demonstrate that there are barriers that restrict luciferin entry into adult plants, and that luciferin availability can be a limiting factor in non-invasive luciferase assays. Received: 11 March 2000 / Accepted: 16 May 2000     

The Role of Superoxide Dismutase in Regulating the Light Emission of Luminescent Fungi

Luminescent fungi spontaneously emit light during certain stagesof their life cycles. Most of them are luminous during a partof their mycelial stage, but not many of them are luminous whenthey form fruiting bodies. In the case of Panellus stipticus,both the mycelium and the fruiting body can be luminous, andthe emission of light takes place when its luciferin is aerobicallyoxidized in the presence of the superoxide anion (O₂) and acationic surfactant. It is highly likely that the luminescencereactions of all kinds of luminous fungi are basically the sameas that of P. stipticus. In order to determine the factor thatmakes a fungus luminous or non-luminous, we studied the relationsbetween the light emission of fungi at various growth stagesand the contents of luciferin, its precursor, superoxide dismutase(SOD), and catalase, on six species of luminescent fungi: Armillariellamellea, Mycena citricolor, Mycena lux-coeli, Omphlotus olearious,Panellus stipticus, and Pleurotus japonicus. The analysis ofthe data suggested that the fungi generally contain the componentsnecessary for light emission, but also contain very large amountsof SOD which destroy O₂^–. If an appreciable amount ofSOD is distributed at the site of light emission, the luminescencereaction is prevented. For the reaction to take place, it isessential that the SOD activity at the site is sufficientlylow or inhibited, despite the high content of SOD in the wholetissue. Thus, the level of SOD activity at the site of lightemission appears to be a limiting factor in regulating the luminescenceof fungi. Key words: Bioluminescence, chemiluminescence, luminous fungi, superoxide ion, superoxide dismutase     

A model considering light reabsorption processes to correct in vivo chlorophyll fluorescence spectra in apples.

Chlorophyll-a contained in the peel of Granny Smith apples emits fluorescence upon excitation with blue light. The observed emission, collected by an external detector and corrected by its spectral response, is still distorted by light reabsorption processes taking place in the fruit skin and differs appreciably from the true spectral distribution of fluorescence emerging from chlorophyll molecules in the biological tissue. Reabsorption processes particularly affect the ratio of fluorescence intensities at 680 nm and at 730 nm. A model to obtain the correct spectral distribution of the emission, from the experimental fluorescence recorded at a fluorometer detector and corrected for the detector spectral sensitivity, is developed in the present work. Measurements of the whole fruit reflectance, the peel transmittance and the flesh reflectance allow the calculation of the reabsorption-corrected spectra. The model is validated by comparing the corrected emission spectra with that obtained for a thin layer of apple-peel-chloroplasts, where no reabsorption takes place. It is recommended to correct distortions in emission spectra of intact fruits due to light reabsorption effects whenever a correlation between the physiological state of the fruit and its fluorescence spectra is investigated.     

Natural animal coloration can Be determined by a nonfluorescent green fluorescent protein homolog

It is generally accepted that the colors displayed by living organisms are determined by low molecular weight pigments or chromoproteins that require a prosthetic group. The exception to this rule is green fluorescent protein (GFP) from Aequorea victoria that forms a fluorophore by self-catalyzed protein backbone modification. Here we found a naturally nonfluorescent homolog of GFP to determine strong purple coloration of tentacles in the sea anemone Anemonia sulcata. Under certain conditions, this novel chromoprotein produces a trace amount of red fluorescence (emission lambda(max) = 595 nm). The fluorescence demonstrates unique behavior: its intensity increases in the presence of green light but is inhibited by blue light. The quantum yield of fluorescence can be enhanced dramatically by single amino acid replacement, which probably restores the ancestral fluorescent state of the protein. Other fluorescent variants of the novel protein have emission peaks that are red-shifted up to 610 nm. They demonstrate that long wavelength fluorescence is attainable in GFP-like fluorescent proteins.     

一种基于LED灯的自适应捕虫方法

害虫对光的敏感波长是随害虫种类、 季节等因素变化而变化, 传统捕虫灯存在发光波长类型较少、 灯与灯之间独立工作不通信的问题, 会造成捕虫灯捕虫有效性低、 能源浪费等问题。为了解决单个捕虫灯发光波长的单一性和多灯独立工作问题, 本研究通过理论分析和相应的系统设计, 得出多个灯捕虫量的最大期望值, 提出了单灯的多波长性实现方法和多灯的协调工作算法。其中单灯的多波长性是基于LED灯多波长性、 低功耗性、 易于维护等性质提出的; 多灯的协调工作算法是指通过中心节点灯与各节点灯的协调通信, 使单灯可自适应控制自身发光波长, 最终使网络中大部分节点灯波长为最佳波长, 小部分节点灯为非最佳波长, 这种方法在实现捕虫高效性的同时, 可实时监测虫种类变化, 达到自适应捕虫方法的最优化。最后通过野外实地试验验证了模拟简化的自适应捕虫方法, 结果证实了本方法在技术上的可行性和高效性。由此使这种LED捕虫灯可以方便地用于山地等复杂的野外环境中, 其中多灯的联合协作工作, 使每个捕虫灯自适应的改变发光波长, 提高了此方法的捕虫效率。