Masakazu Katsumata

New feature of delayed luminescence: preillumination-induced concavity and convexity in delayed luminescence decay curve in the green alga Pseudokirchneriella subcapitata.

A new method for measuring delayed luminescence (delayed fluorescence) employs preillumination and a dark waiting period before normal excitation. The preillumination results in a concavity and a convexity in the decay curve in delayed luminescence in the green alga Pseudokirchneriella subcapitata. Formation of the concavity and the convexity is not affected by excitation wavelength (680 nm and 700 nm). However, the concavity and the convexity progressively decrease as the dark waiting period increases after preillumination. The formation of the concavity and the convexity was inhibited by exposure to the electron transport inhibitors DBMIB (644 microg/L, 2.0 microM) and Antimycin A (55 microg/L, 0.1 microM). Samples exposed to DBMIB exhibited noticeable reduction in the concavity, whereas samples exposed to Antimycin A exhibited pronounced reduction in the convexity. There is a possibility that the formation and disappearance of the concavity and the convexity are due to the reduction-oxidation state of the plastoquinone pool and the cyclic electron transport. We expect this method being useful in evaluating the effects of chemicals (particularly toxic chemicals) on photosynthetic reactions, and the method may also help to resolve questions regarding the source of long delayed luminescence.

Lunisolar tidal synchronism with biophoton emission during intercontinental wheat-seedling germination tests

Synchronic measurements of spontaneous ultra-weak light emission from germinating wheat seedlings both in Brazil and after transportation to Japan, and with a simultaneous series of germinations with local seedlings in the Czech Republic, are presented. A series of tests was also performed with samples returned from Japan to Brazil and results compared with those from undisturbed Brazilian seedlings. Native seedlings presented semi-circadian rhythms of emission which correlated with the gravimetric tidal acceleration at their locality, as did seeds which had been transported from Brazil to Japan, and then returned to Brazil. Here, however, there were very small disturbances within the periodicity of emissions, perhaps as a result of similar tidal profiles at locations whose longitudes are 180° apart, as in this case, different from previous results obtained in Brazil–Germany tests with other longitude shift. This feature of the Brazil and Japan locations may have minimized the requirement for the acclimatization of the transported seed to their new location.

Simultaneous monitoring of superoxides and intracellular calcium ions in neutrophils by chemiluminescence and fluorescence: Evaluation of action mechanisms of bioactive compounds in foods

We have developed a measuring system for simultaneous monitoring of chemiluminescence and fluorescence, which indicate respectively, (i) generation of superoxide anion radicals (O2(-•)) and (ii) change in the intracellular calcium ion concentration ([Ca(2+)]i) of neutrophils triggered by the mechanism of innate immune response. We applied this measuring system for establishing a method to distinguish between anti-inflammatory actions and antioxidant actions caused by bioactive compounds. We evaluated anti-inflammatory agents (zinc ion [Zn(2+)] and ibuprofen) and antioxidants (superoxide dismutase [SOD] and ascorbic acid). It was shown that ibuprofen and Zn(2+) were anti-inflammatory while SOD and ascorbic acid were anti-oxidative. We conclude that it is possible to determine the mechanism of action of bioactive compounds using this method.

Rapid ecotoxicological bioassay using delayed fluorescence in the marine cyanobacterium Cyanobium sp. (NIES-981)

The use of delayed fluorescence intensity as an endpoint for rapid estimation of the effective concentration (ECx) has been reported as an alternative to standard growth inhibition (at 72 h after exposure) in some algal species including Pseudokirchneriella subcapitata. In marine algae, although an approach of bioassaying using delayed fluorescence measurements has not been performed yet, its development would provide many benefits for marine environmental risk assessment. In this study, we selected marine cyanobacterium Cyanobium sp. (NIES-981) as our test algal species and demonstrated that this species is valid for the standard growth inhibition test based on criteria provide by Organization for Economic Co-operation and Development guidelines. Furthermore, standard inhibition tests and shorter period test using DF were performed in NIES-981 using five chemicals (3,5-DCP, simazine, diflufenican, K2Cr2O7, and CuSO4), and their EC50 and low-toxic-effect values (EC10, EC5, and NOEC) were determined from two dose-response curves. Based on comparisons of the two dose-response curves and the EC50 values, we conclude that DF intensity is useful as an endpoint for rapid estimation of EC50 in NIES-981.

Validation of rapid algal bioassay using delayed fluorescence in an interlaboratory ring study

Algal growth inhibition tests are generally used to determine the toxic effects of chemical substances on algae growth. In this report, we describe a rapid and simple test procedure using delayed fluorescence (DF) to determine chemical toxicities more rapidly than the conventional 72h or 96h growth inhibition tests. We assess the suitability of DF to serve as an alternative endpoint for biomass production and determine the variability by an interlaboratory ring study using a typical reference toxicant 3,5-dichlorophenol (DCP). The results suggest that DF has the potential to be used as a surrogate measure of photosynthetically-active biomass in the algal growth inhibition tests. The half maximal effective concentration (EC50) values of DCP determined from the DF inhibition test in 6h and 24h (1.2±0.3mg/L and 2.7±0.5mg/L respectively) are in reasonable agreement with the EC50 value of DCP determined by the 72h conventional method (1.8mg/L). In the interlaboratory ring study, the intralaboratory and interlaboratory variabilities of the EC50 of the DF inhibition test for a 24h exposure period are 12% and 28% respectively. DF intensity can be considered as a surrogate of living biomass with active photosynthesis, and we conclude that a 24h exposure duration better estimates the toxic effects measured using conventional surrogate measures for dry weight such as cell counts, volume, optical density or fluorescence.

Delayed fluorescence as an indicator of the influence of the herbicides Irgarol 1051 and Diuron on hard coral Acropora digitifera

We examined the effect of two herbicides (Irgarol 1051 and Diuron) on symbiotic dinoflagellates in the hard coral Acropora digitifera using delayed fluorescence (DF), specifically assessing changes in molecular membrane transport, i.e. inflow and outflow rates, and the binding of the herbicides to target proteins in photosystem II. The DF approach is rapid (e.g. measurement time, 60 s) and non-invasive, and can provide data on the extent of a photosynthetic system and the activity of its electron carriers. The DF of A. digitifera is inhibited 2 h after exposure to 1 μg/L of either Irgarol or Diuron. Analysis of DF inhibition over time by a compartment model suggests that Irgarol exposure results in a relatively higher inflow rate and lower outflow rate than does Diuron exposure. This suggests that Irgarol exposure more strongly inhibits photosynthesis and that the coral symbiotic dinoflagellates recover less from inhibition.

Nondestructive evaluation of photosynthesis by delayed luminescence in Arabidopsis in Petri dishes

Nondestructive evaluation of photosynthesis is a valuable tool in the field and laboratory. Delayed luminescence (DL) can reflect charge recombination through the backflow of electrons. However, DL detection has not yet been adapted for whole plants in Petri dishes. To compensate for differences in DL decay between sibling Arabidopsis plants grown under the same conditions, we developed a time-sequential double measurement method. Using this method, we examined the influence of photosynthetic electron flow inhibitors, and differences in the DL decay curves were categorized by considering the initial and late phases of the decay curves, as well as their intermediate slopes. The appearance of concavity and convexity in DL curves in Arabidopsis was different from unicellular algae, suggesting complexity in the photosynthetic machinery of higher plants. This detection method should be invaluable for evaluating photosynthetic defects in higher plants under sterile conditions without interrupting plant culture. Graphical abstract Nondestructive evaluation of photosynthesis by delayed luminescence (DL) in Arabidopsis with time-sequential double measurement to compensate for individual differences.

Fluorescence decay of dyed protozoa: differences between stressed and non-stressed cysts

Several series of tests have shown that fresh, intact samples of Giardia duodenalis and Cryptosporidium parvum (oo)cysts are not marked by fluorescent probes such as carboxyfluorcein-succinimidyl-diacetate-ester (CFDA-SE), C12-resazurin and SYTOX® Green, probably because of their robust cell walls. These dyes fail to indicate the viability of such protozoa and allow negative responses to be recorded from living and infectious samples. Cryptosporidium parvum showed stronger isolation from chemicals, with living oocysts remaining unstained by the probe for up to 90 days after extraction. However, in further fluorescence decay (FD) experiments run with G. duodenalis samples stained using CFDA-SE (comprising living, non-stressed but aged cysts, heat-killed samples and UV-C-stressed samples) each showed a different FD decay profile, here studied in seven series of tests of five replicates each. The FD profiles were fitted by double-exponential decay kinetics, with the decay constant k2 being five times higher than k1. This FD procedure is fast and can be easily reproduced in 10 steps, taking ~ 1 h of laboratory work for already purified samples.

Evaluation of the toxicity of leaches from hydrothermal sulfide deposits by means of a delayed fluorescence-based bioassay with the marine cyanobacterium Cyanobium sp. NIES-981

The commercial use of metals such as copper, lead, and zinc has markedly increased in recent years, resulting in increased interest in deep-sea mining of seafloor hydrothermal sulfide deposits. However, the full extent of the impact of deep-sea mining at hydrothermal field deposits on the environment remains unclear. In addition to impacting the deep sea, the leaching of heavy metals from extracted sulfide mineral may also affect the upper ocean zones as the sulfide rock is retrieved from the seafloor. Here, we used a delayed fluorescence-based bioassay using the marine cyanobacterium Cyanobium sp. NIES-981 to evaluate the toxicity of three sulfide core samples obtained from three drill holes at the Izena Hole, middle Okinawa Trough, East China Sea. Leaches from two of the cores contained high concentrations of zinc and lead, and they markedly inhibited delayed fluorescence in Cyanobium sp. NIES-981 compared with control. By examining the toxicity of artificial mixed-metal solutions with metal compositions similar to those of the leaches, we confirmed that this inhibition was a result of high zinc and lead concentrations into the leaches. In addition, we conclude that this delayed fluorescence-based bioassay is a viable method for use by deep-sea mining operations because it is quicker and requires less laboratory space and equipment than the standard assay.