Photobiological Experiments Research Articles

Photo-biological Effectiveness of PAR for Radish Plant Communities Irradiated Using an LED Source with an Adjustable Spectrum

The aim of the current study was to find the most effective spectral modes of irradiation of short-season plants (for example, radish) using a fluorescent LED irradiator with a variable spectrum. The research methodology was based on the species-specific response of plants to the spectral composition of light. Photo-biological experiments were carried out to find effective spectral composition of PAR for cultivating radish communities under stable and variable irradiation modes of electrical lighting. It was shown that under stable irradiation modes, the highest productivity of economically useful radish biomass had been achieved at spectral composition with a maximum of 60 % radiation in the (600–700) nm region in a variable spectral irradiation mode. The variable spectral irradiation mode turned out to be the most effective when the spectral composition with the dominance of red rays was changed to the spectral composition with the dominance of blue rays. There were no significant differences between the best production parameters for stable and variable modes of irradiation of radish plants, which indicates the expediency of choosing the stable spectral mode of irradiation of radish plants as a short-season crop under electrical illumination. The results obtained can be used to select spectral irradiation modes for short-season plants grown under electrical lighting in northern regions and in isolated rooms in various climatic zones using “City-farm” technologies.

First report of trans-A2B-corrole derived from a lapachone derivative: photophysical, TD-DFT and photobiological assays.

In this work, the synthesis, characterization and photophysical assays of a new trans-A2B-corrole derivative from the naturally occurring quinone are described. β-Lapachone is a naturally occurring quinoidal compound that provides highly fluorescent heterocyclic compounds such as lapimidazoles. The new trans-A2B-corrole compound was obtained from the reaction between 2,3,4,5,6-(pentafluorophenyl)dipyrromethane and the lapimidazole bearing an aldehyde group. The dyad was characterized by high-resolution mass spectrometry (HRMS), NMR spectroscopy (1H, COSY 2D, HMBC, 19F), FT-IR, UV-vis, steady-state and time-resolved fluorescence, electrochemical studies (CV), TD-DFT analysis and photobiological experiments, in which includes aggregation, stability in solution, photostability and partition coefficients assays. Finally, ROS generation assays were performed using 1,3-diphenylisobenzofuran (DPBF) method and the presented compound showed significant photostability and singlet oxygen production.

Photochemical defense as trait of fungi from Cortinarius subgenus Dermocybe

The photobiological activity of ten colorful species belonging to subgenus Dermocybe of the basidiomycete genus Cortinarius was investigated. Extracts of all species produced singlet oxygen and are thus photoactive. Pigment analysis was performed and showed similarities of the anthraquinone pigments across the species in dependency to their respective pigmentation types. Detailed content analysis of the pigments in the whole agaricoid fruiting body compared to the three different tissue types (pileus, stipe, and lamellae) revealed that the pigments emodin, dermocybin, and dermorubin, as well as their respective glycosides, are enhanced in the gills. In an independent experiment, the gills were shown to be the most photoactive tissues of the fruiting body. Photobiological experiments with invertebrates (i.e., glassworm Chaoborus crystallinus) proved a phototoxic effect of the methanolic extract of the red blood webcap (Cortinarius sanguineus var. aurantiovaginatus). This work adds further evidence to a common photobiological trait in Cortinarius subgenus Dermocybe and underpins the possibility of a photochemical defense mechanism in fungi.Graphical abstract

Toxicity of UV filters on marine bacteria: Combined effects with damaging solar radiation

Organic UV filters are of emerging concern due to their occurrence and persistence in coastal ecosystems. Because marine bacteria are crucial in the major biogeochemical cycles, there is an urgent need to understand to what extent these microorganisms are affected by those chemicals. This study deciphers the impact of five common sunscreen UV filters on twenty-seven marine bacteria, combining both photobiology and toxicity analysis on environmentally relevant species. Seven bacteria were sensitive to different organic UV filters at 1000 μg L−1, including octinoxate and oxybenzone. This is the first report demonstrating inhibition of bacterial growth from 100 μg L−1. None of the UV filters showed any toxicity at 1000 μg L−1 on stationary phase cells, demonstrating that physiological state was found to be a key parameter in the bacterial response to UV-filters. Indeed, non-growing bacteria were resistant to UV filters whereas growing cells exhibited UV filter dependent sensitivity. Octinoxate was the most toxic chemical at 1000 μg L−1 on growing cells. Interestingly, photobiology experiments revealed that the toxicity of octinoxate and homosalate decreased after light exposure while the other compounds were not affected. In terms of environmental risk characterization, our results revealed that the increasing use of sun blockers could have detrimental impacts on bacterioplanktonic communities in coastal areas. Our findings contribute to a better understanding of the impact of the most common UV filters on bacterial species and corroborate the importance to consider environmental parameters such as solar radiation in ecotoxicology studies.

Optimisation of Lighting Parameters of Irradiation in Light Culture of Lettuce Plants Using LED Emitters

The results of the ending phase of photo biological studies of capacity of lettuce and basil grown in conditions of a phytotron with irradiation by radiation of different ratio of fractions in the red-blue and blue-green-red regions of photosynthetic active radiation (PAR) are presented. The spectral variants of PAR providing the maximum or near to maximum capacity of the specified cultures are found. The complex and multiple-valued nature of effect of main PAR spectral regions on photo energetic and photo regulatory processes in plants providing their capacity is highlighted, which makes it impossible to determine common unified requirements to optimal parameters of artificial irradiation for growing of plants. These requirements shall be defined on the basis of direct photo biological experiments with main species of agricultural plants and be the most important element of the general photo culture macro technology. The general principles of fulfilment of the specified requirements to PAR spectra of phytoemitters are reviewed, including with consideration of setting of normal vision conditions for personnel of greenhouses and other protected ground structures.

Design and validation of an open-source modular Microplate Photoirradiation System for high-throughput photobiology experiments.

Research in photobiology is currently limited by a lack of devices capable of delivering precise and tunable irradiation to cells in a high-throughput format. This limits researchers to using expensive commercially available or custom-built light sources which make it difficult to replicate, standardize, optimize, and scale experiments. Here we present an open-source Microplate Photoirradiation System (MPS) developed to enable high-throughput light experiments in standard 96 and 24-well microplates for a variety of applications in photobiology research. This open-source system features 96 independently controlled LEDs (4 LEDs per well in 24-well), Wi-Fi connected control and programmable graphical user interface (GUI) for control and programming, automated calibration GUI, and modular control and LED boards for maximum flexibility. A web-based GUI generates light program files containing irradiation parameters for groups of LEDs. These parameters are then uploaded wirelessly, stored and used on the MPS to run photoirradiation experiments inside any incubator. A rapid and semi-quantitative porphyrin metabolism assay was also developed to validate the system in wild-type fibroblasts. Protoporphyrin IX (PpIX) fluorescence accumulation was induced by incubation with 5-aminolevulinic acid (ALA), a photosensitization method leveraged clinically to destroy malignant cell types in a process termed photodynamic therapy (PDT), and cells were irradiated with 405nm light with varying irradiance, duration and pulsation parameters. Immediately after light treatment with the MPS, subsequent photobleaching was measured in live, adherent cells in both 96-well and a 24-well microplates using a microplate reader. Results demonstrate the utility and reliability of the Microplate Photoirradiation System to irradiate cells with precise irradiance and timing parameters in order to measure PpIx photobleaching kinetics in live adherent cells and perform comparable experiments with both 24 and 96 well microplate formats. The high-throughput capability of the MPS enabled measurement of enough irradiance conditions in a single microplate to fit PpIX fluorescence to a bioexponential decay model of photobleaching, as well as reveal a dependency of photobleaching on duty-cycle—but not frequency—in a pulsed irradiance regimen.

An open-hardware platform for optogenetics and photobiology.

In optogenetics, researchers use light and genetically encoded photoreceptors to control biological processes with unmatched precision. However, outside of neuroscience, the impact of optogenetics has been limited by a lack of user-friendly, flexible, accessible hardware. Here, we engineer the Light Plate Apparatus (LPA), a device that can deliver two independent 310 to 1550 nm light signals to each well of a 24-well plate with intensity control over three orders of magnitude and millisecond resolution. Signals are programmed using an intuitive web tool named Iris. All components can be purchased for under $400 and the device can be assembled and calibrated by a non-expert in one day. We use the LPA to precisely control gene expression from blue, green, and red light responsive optogenetic tools in bacteria, yeast, and mammalian cells and simplify the entrainment of cyanobacterial circadian rhythm. The LPA dramatically reduces the entry barrier to optogenetics and photobiology experiments.

Comparison of the acute ultraviolet photoresponse in congenic albino hairless C57BL/6J mice relative to outbred SKH1 hairless mice

Hairless albino Crl:SKH1-Hr(hr) mice are commonly utilized for studies in which hair or pigmentation would introduce an impediment to observational studies. Being an outbred strain, the SKH1 model suffers from key limitations that are not seen with congenic mouse strains. Inbred and congenic C57BL/6J mice are commonly utilized for modified genetic mouse models. We compare the acute UV-induced photoresponse between outbred SKH1 mice and an immune competent, hairless, albino C57BL/6J congenic mouse line [B6.Cg-Tyr(c-2J) Hr(hr) /J]. Histologically, B6.Cg-Tyr(c-2J) Hr(hr) /J skin is indistinguishable from that of SKH1 mice. The skin of both SKH1 and B6.Cg-Tyr(c-2J) Hr(hr) /J mice exhibited a reduction in hypodermal adipose tissue, the presence of utricles and dermal cystic structures, the presence of dermal granulomas and epidermal thickening. In response to a single 1500 J/m(2) ultraviolet B dose, the oedema and apoptotic responses were equivalent in both mouse strains. However, B6.Cg-Tyr(c-2J) Hr(hr) /J mice exhibited a more robust delayed sunburn reaction, with an increase in epidermal erosion, scab formation and myeloperoxidase activity relative to SKH1 mice. Compared with SKH1 mice, B6.Cg-Tyr(c-2J) Hr(hr) /J also exhibited an aberrant proliferative response to this single UV exposure. Epidermal Ki67 immunopositivity was significantly suppressed in B6.Cg-Tyr(c-2J) Hr(hr) /J mice at 24 h post-UV. A smaller non-significant reduction in Ki67 labelling was observed in SKH1 mice. Finally, at 72 h post-UV, SKH1 mice, but not B6.Cg-Tyr(c-2J) Hr(hr) /J mice, exhibited a significant increase in Ki67 immunolabelling relative to non-irradiated controls. Thus, B6.Cg-Tyr(c-2J) Hr(hr) /J mice are suitable for photobiology experiments.

UV SURFACE ENVIRONMENT OF EARTH-LIKE PLANETS ORBITING FGKM STARS THROUGH GEOLOGICAL EVOLUTION

The UV environment of a host star affects the photochemistry in the atmosphere, and ultimately the surface UV environment for terrestrial planets and therefore the conditions for the origin and evolution of life. We model the surface UV radiation environment for Earth-sized planets orbiting FGKM stars at the 1AU equivalent distance for Earth through its geological evolution. We explore four different types of atmospheres corresponding to an early Earth atmosphere at 3.9 Gyr ago and three atmospheres covering the rise of oxygen to present day levels at 2.0 Gyr ago, 0.8 Gyr ago and modern Earth (Following Kaltenegger et al. 2007). In addition to calculating the UV flux on the surface of the planet, we model the biologically effective irradiance, using DNA damage as a proxy for biological damage. We find that a pre-biotic Earth (3.9 Gyr ago) orbiting an F0V star receives 6 times the biologically effective radiation as around the early Sun and 3520 times the modern Earth-Sun levels. A pre-biotic Earth orbiting GJ 581 (M3.5V) receives 300 times less biologically effective radiation, about 2 times modern Earth-Sun levels. The UV fluxes calculated here provide a grid of model UV environments during the evolution of an Earth-like planet orbiting a range of stars. These models can be used as inputs into photo-biological experiments and for pre-biotic chemistry and early life evolution experiments.

Regression Model for Estimating Inactivation of Microbial Aerosols by Solar Radiation

The inactivation of pathogenic aerosols by solar radiation is relevant to public health and biodefense. We investigated whether a relatively simple method to calculate solar diffuse and total irradiances could be developed and used in environmental photobiology estimations instead of complex atmospheric radiative transfer computer programs. The second-order regression model that we developed reproduced 13 radiation quantities calculated for equinoxes and solstices at 35(°) latitude with a computer-intensive and rather complex atmospheric radiative transfer program (MODTRAN) with a mean error <6% (2% for most radiation quantities). Extending the application of the regression model from a reference latitude and date (chosen as 35° latitude for 21 March) to different latitudes and days of the year was accomplished with variable success: usually with a mean error <15% (but as high as 150% for some combination of latitudes and days of year). This accuracy of the methodology proposed here compares favorably to photobiological experiments where the microbial survival is usually measured with an accuracy no better than ±0.5 log10 units. The approach and equations presented in this study should assist in estimating the maximum time during which microbial pathogens remain infectious after accidental or intentional aerosolization in open environments.

Phosphorylation by CK2 Enhances the Rapid Light-induced Degradation of Phytochrome Interacting Factor 1 in Arabidopsis

The phytochrome family of sensory photoreceptors interacts with phytochrome interacting factors (PIFs), repressors of photomorphogenesis, in response to environmental light signals and induces rapid phosphorylation and degradation of PIFs to promote photomorphogenesis. However, the kinase that phosphorylates PIFs is still unknown. Here we show that CK2 directly phosphorylates PIF1 at multiple sites. α1 and α2 subunits individually phosphorylated PIF1 weakly in vitro. However, each of four β subunits strongly stimulated phosphorylation of PIF1 by α1 or α2. Mapping of the phosphorylation sites identified seven Ser/Thr residues scattered throughout PIF1. Ser/Thr to Ala scanning mutations at all seven sites eliminated CK2-mediated phosphorylation of PIF1 in vitro. Moreover, the rate of degradation of the Ser/Thr to Ala mutant PIF1 was significantly reduced compared with wild-type PIF1 in transgenic plants. In addition, hypocotyl lengths of the mutant PIF1 transgenic plants were much longer than the wild-type PIF1 transgenic plants under light, suggesting that the mutant PIF1 is suppressing photomorphogenesis. Taken together, these data suggest that CK2-mediated phosphorylation enhances the light-induced degradation of PIF1 to promote photomorphogenesis.

Chemical “light meters” for photochemical and photobiological studies

Nitrate and nitrite solar actinometers or chemical ‘light meters’ were used to quantify light doses in photochemical and photobiological experiments involving dimethylsulfide (DMS) and dimethylsulfoniopropionate (DMSP) cycling. Light doses were calculated based on the photochemical production of salicylic acid (SA) from benzoic acid in these actinometers, with SA quantified by either spectrofluorometry or high performance liquid chromatography. Nitrate and nitrite actinometers were modified for deployment at low temperatures in Antarctic waters by addition of sodium chloride as a freezing point depressant. The addition of salt did not affect the solar response of the actinometers; however, the solar response did change slightly with latitude. In the Antarctic, peak response wavelengths (and bandwidths) for the Mylar D-wrapped actinometers in quartz tubing were 326 nm (319 – 333 nm) and 353 nm (325 – 380 nm) for nitrate and nitrite, respectively, and these were 2 – 5 nm blue shifted compared to the peak response wavelengths and bandwidths observed in the Sargasso Sea. Excellent agreement was observed when comparing the integrated irradiance determined with the actinometers to that determined with a spectroradiometer. Likewise, diffuse attenuation coefficients for downwelling irradiance (Kd(λ)) calculated from water column actinometer measurements agreed well with Kd(λ) values calculated from irradiance measurements determined with a Biospherical PUV-511 profiling radiometer. Actinometers were used to measure light doses in experiments involving DMS and DMSP transformations during several field campaigns in the Ross Sea, Antarctica and the Sargasso Sea. Based on actinometer measurements, it was determined that DMS photolysis was dependent on UV irradiation between approximately 325 – 380 nm, while biological consumption rates of DMS and DMSP were inhibited by radiation at wavelengths less than approximately 333 nm. When DMS photolysis rate constants were expressed in terms of light dose rather than time, it was possible to 1) directly determine photolysis rate constants in the water column and 2) directly compare photolysis rate constants across diverse oceanographic regions.

PHYTOCHROME KINASE SUBSTRATE 1 is a phototropin 1 binding protein required for phototropism

Phototropism, or plant growth in response to unidirectional light, is an adaptive response of crucial importance. Lateral differences in low fluence rates of blue light are detected by phototropin 1 (phot1) in Arabidopsis. Only NONPHOTOTROPIC HYPOCOTYL 3 (NPH3) and root phototropism 2, both belonging to the same family of proteins, have been previously identified as phototropin-interacting signal transducers involved in phototropism. PHYTOCHROME KINASE SUBSTRATE (PKS) 1 and PKS2 are two phytochrome signaling components belonging to a small gene family in Arabidopsis (PKS1-PKS4). The strong enhancement of PKS1 expression by blue light and its light induction in the elongation zone of the hypocotyl prompted us to study the function of this gene family during phototropism. Photobiological experiments show that the PKS proteins are critical for hypocotyl phototropism. Furthermore, PKS1 interacts with phot1 and NPH3 in vivo at the plasma membrane and in vitro, indicating that the PKS proteins may function directly with phot1 and NPH3 to mediate phototropism. The phytochromes are known to influence phototropism but the mechanism involved is still unclear. We show that PKS1 induction by a pulse of blue light is phytochrome A-dependent, suggesting that the PKS proteins may provide a molecular link between these two photoreceptor families.

COMPARISON OF THE SPECTRAL DISTRIBUTION OF ARTIFICIAL ULTRAVIOLET SOURCES WITH SOLAR RADIATION FOR PHOTOBIOLOCICAL EXPERIMENTS

For photochemical and photobiological experiments there is a great need to mimic the spectral distribution of solar ultraviolet radiation. The spectral distribution of the solar simulated radiation has to be compared with a reference solar spectrum. Therefore a integral measure over the whole spectral range was developed to check the spectral fit. This index of the spectral fit ISF is calculated by the relative irradiance which is normalized to unity in the way that the integral of the spectral irradiance over the wavelengths gives one. The index gives the portion of the area under the relative spectral irradiance of the solar simulated radiation and the and reference spectrum of the sun.

Common Fluorescent Sunlamps are an Inappropriate Substitute for Sunlight¶

Fluorescent sunlamps are commonly employed as convenient sources in photobiology experiments. The ability of Kodacel to filter photobiologically irrelevant UVC wavelengths has been described. Yet there still remains a major unaddressed issue--the over representation of UVB in the output. The shortest terrestrial solar wavelengths reaching the surface are approximately 295 nm with the 295-320 nm range comprising approximately 4% of the solar UV irradiance. In Kodacel-filtered sunlamps, 47% of the UV output falls in this range. Consequently, in studies designed to understand skin photobiology after solar exposure, the use of these unfiltered sunlamps may result in misleading, or even incorrect conclusions. To demonstrate the importance of using an accurate representation of the UV portion of sunlight, the ability of different ultraviolet radiation (UVR) sources to induce the expression of a reporter gene was assayed. Unfiltered fluorescent sunlamps (FS lamps) induce optimal chloramphenicol acetyltransferase (CAT) activity at apparently low doses (10-20 J/cm2). Filtering the FS lamps with Kodacel raised the delivered dose for optimal CAT activity to 50-60 mJ/cm2. With the more solar-like UVA-340 lamps somewhat lower levels of CAT activities were induced even though the apparent delivered doses were significantly greater than for either the FS or Kodacel-filtered sunlamp (KFS lamps). When DNA from parallel-treated cells was analyzed for photoproduct formation by a radioimmuneassay, it was shown that the induction of CAT activity correlated with the level of induced photoproduct formation regardless of the source employed.

Dihydrothienopsoralens: New furocoumarins as potential photoreacting agents

A series of new dihydrothienopsoralen derivatives were synthesized from 6-hydroxybenzo[b]furan in four steps in good yield. These compounds were prepared as potential photosensitizing and chemotherapeutic agents for psoriasis. The spectroscopy study in also reported and showed an absorbance at λ = 364 nm and fluorescence at λ = 440 nm with high molar absorptivity, wave length used to perform the photochemical and photobiological experiments.

Biologically active insolation over Antarctic waters: Effect of a highly reflecting coastline

Near an Antarctic coastline or sea ice edge, multiple reflection of photons between the high‐albedo surface and a cloud will increase the downwelling surface insolation not only over the high‐albedo surface itself but also out over the adjacent open water. This insolation enhancement is examined with a Monte Carlo radiative transfer model. The insolation enhancement extends to a typical distance of 4 km out to sea, with the most important effects being within 2 km of the coastline. The strength of the multiple reflection effect depends primarily on cloud base height and cloud optical depth and only slightly on cloud geometrical thickness. The insolation enhancement is also a function of wavelength, being larger for ultraviolet wavelengths than for the visible. This is due to a slightly greater contribution from Rayleigh scattering at the shorter wavelengths, although at ultraviolet wavelengths where ozone absorption is strong, tropospheric ozone absorption can offset the Rayleigh scattering contribution at larger cloud optical depths. On the basis of the limited range of the multiple reflection effect (2–4 km out to sea) the insolation enhancement due to the high‐albedo coastline is unlikely to be a major influence on the primary productivity of all Antarctic waters; however, it may influence phytoplankton blooms near the coast and photobiological experiments carried out at coastal research stations. Also, the insolation enhancement may have significance in sea ice leads and polynyas.

Gametic Differentiation of Chlamydomonas reinhardtii

Gametic differentiation of the unicellular green alga Chlamydomonas reinhardtii proceeds in two steps controlled by the extrinsic signals nitrogen deficiency and light. Nitrogen deprivation induces the differentiation of vegetative cells to sexually immature pregametes. A light signal is required to convert the pregametes to gametes. Both signals are also required for the maintenance of mating competence. Two converging signal transduction chains are proposed to control gamete formation. For the differentiation of pregametes to gametes, a fluence rate-dependent reaction, requiring continuous irradiation, is suggested by photobiological experiments.

46] Techniques for studying photoprotective function of carotenoid pigments

This chapter describes how to perform experiments in various kinds of organisms to study the ability of carotenoid pigments to protect against photosensitization. These techniques can be used to determine if carotenoids of interest can give protection, as well as to study the mechanisms of protection. Carotenoid protection against photosensitization by either endogenous or exogenous photosensitizers can be easily studied in bacteria. For photobiological experiments to determine the protective ability of carotenoids, cultures of the wild type and mutant(s) to be studied are harvested in the logarithmic phase of growth, centrifuged, and resuspended in phosphate or Tris buffer to contain about 10 6 to 10 7 cells/ml. As far as mammals are concerned, various kinds of fibroblasts, including mammary cells and erythrocytes from protoporphyric patients have been studied; the former have been used to determine the ability of carotenoids to prevent various kinds of cellular and nuclear damage, and the latter to see if carotenoids can prevent photohemolysis. Continued studies of this most important function of carotenoids are well warranted.

Free-electron lasers in ultraviolet photobiology

The potential uses for a free-electron laser (FEL), tunable in wavelength from 10 to 400 nm, for photobiological experiments is discussed. Inherent problems of cell and molecular absorption, especially in certain regions of the ultraviolet (UV), are addressed. Absorption values for living cells and viruses at selected wavelengths in the UV are tabulated, and a calculation of the flux needed to inactivate mammalian cells is included. A comparison is made of the UV output of a proposed rf-linac FEL with those of a monochromator, a tunable dye laser, and a synchrotron. The advantages of a UV FEL are apparent, especially in the wavelength regions where the cross section for absorption by biological molecules is low, i.e., 300 to 400 nm and 10 to 200 nm. It is apparent that a UV FEL would be an ideal source for a variety of biological studies that use both intact organisms and isolated cells and viruses.