Liquid-microjet photoelectron spectroscopy of the green fluorescent protein chromophore.

Green fluorescent protein (GFP), the most widely used fluorescent protein for in vivo monitoring of biological processes, is known to undergo photooxidation reactions. However, the most fundamental property underpinning photooxidation, the electron detachment energy, has only been measured for the deprotonated GFP chromophore in the gas phase. Here, we use multiphoton ultraviolet photoelectron spectroscopy in a liquid-microjet and high-level quantum chemistry calculations to determine the electron detachment energy of the GFP chromophore in aqueous solution. The aqueous environment is found to raise the detachment energy by around 4 eV compared to the gas phase, similar to calculations of the chromophore in its native protein environment. In most cases, electron detachment is found to occur resonantly through electronically excited states of the chromophore, highlighting their importance in photo-induced electron transfer processes in the condensed phase. Our results suggest that the photooxidation properties of the GFP chromophore in an aqueous environment will be similar to those in the protein.

Recommendations for Reporting Methods in Phototesting Studies.

The figure presented here illuminates the large number of variables that are necessary to adequately describe phototesting protocols. Each of these characteristics can be critical in understanding results presented in the photomedicine community as well as the broader photobiology and photochemistry communities. The inclusion of all of these variables within each phototesting publication will aid in discourse and further scientific discovery within our field.

In search of blue-light effects on cognitive control.

People are constantly exposed to blue light while engaging in work. It is thus crucial to understand if vast exposure to blue light influences cognitive control, which is essential for working efficiently. Previous studies proposed that the stimulation of intrinsically photosensitive retinal ganglion cells (ipRGCs), a newly discovered photoreceptor that is highly sensitive to blue light, could modulate non-image forming functions. Despite studies that showed blue light (or ipRGCs) enhances brain activations in regions related to cognitive control, how exposure to blue light changes our cognitive control behaviorally remains elusive. We examined whether blue light influences cognitive control through three behavioral tasks in three studies: the sustained attention to response task (SART), the task-switching paradigm, and the Stroop task. Classic effects of the SART, switch cost, and the Stroop effect were found, but no differences were observed in results of different background lights across the six experiments. Together, we conclude that these domains of cognitive control are not influenced by blue light and ipRGCs, and whether the enhancement of blue light on brain activities extends to the behavioral level should be carefully re-examined.

New understanding of the direct effects of spectral balance on behaviour in Myzus persicae.

The study of insect responses to colour has mainly focused on flying species and morphs, however colour cues are likely to be important for insect positioning within the canopy. We examine the role of illumination colour in canopy positioning of apterous Myzus persicae (Sulzer) using both a field experiment, utilising various UV-manipulating optical filters, and a laboratory experiment using video tracking of individuals illuminated by a variable intensity UVA-Blue-Green LED-array. In the field experiment, approximately twice as many aphids were located on exposed leaf surfaces under UV-deficient environments compared to UV-rich environments. The lab experiment showed all three M. persicae photoreceptors were involved in a visually-mediated feeding/avoidance behaviour. Highly UV-rich, green-deficient environments were up to 3 times as likely to trigger an avoidance behaviour compared to UV-absent, green-rich environments such as those found below the leaf surface. We show that apterous M. persicae use this, in addition to other cues, in order to locate feeding positions that minimise exposure to direct sunlight. This has relevance to both the fundamental understanding of photoprotective behaviour in Hemiptera as well as to applied research of crop production environments that disrupt pest behaviour.

Evolution-inspired design of multicolored photoswitches from a single cyanobacteriochrome scaffold.

Cyanobacteriochromes (CBCRs) are small, bistable linear tetrapyrrole (bilin)-binding light sensors which are typically found as modular components in multidomain cyanobacterial signaling proteins. The CBCR family has been categorized into many lineages that roughly correlate with their spectral diversity, but CBCRs possessing a conserved DXCF motif are found in multiple lineages. DXCF CBCRs typically possess two conserved Cys residues: a first Cys that remains ligated to the bilin chromophore and a second Cys found in the DXCF motif. The second Cys often forms a second thioether linkage, providing a mechanism to sense blue and violet light. DXCF CBCRs have been described with blue/green, blue/orange, blue/teal, and green/teal photocycles, and the molecular basis for some of this spectral diversity has been well established. We here characterize AM1_1499g1, an atypical DXCF CBCR that lacks the second cysteine residue and exhibits an orange/green photocycle. Based on prior studies of CBCR spectral tuning, we have successfully engineered seven AM1_1499g1 variants that exhibit robust yellow/teal, green/teal, blue/teal, orange/yellow, yellow/green, green/green, and blue/green photocycles. The remarkable spectral diversity generated by modification of a single CBCR provides a good template for multiplexing synthetic photobiology systems within the same cellular context, thereby bypassing the time-consuming empirical optimization process needed for multiple probes with different protein scaffolds.

Method for Investigation of Photobiological Effects of Light on Human Skin Cells Mediated by Low Doses of Light.

Driven by evolution, human skin cells have developed an extraordinary ability both to sense and to respond to the photons of sunlight through a plethora of photobiological interactions, activating intracellular signalling cascades and regulating skin cells homeostasis. It has recently been reported that some of these photobiological responses triggered by low levels of light (or the so-called photobiomodulation) could initiate beneficial therapeutic effects. Identification of these effective light-based therapeutic solutions requires in-depth understanding of the parameter space. The physical, biological, and chemical conditions that need to be fulfilled to facilitate such positive photobiological effects are to be carefully deciphered. Here, we provide the protocols that were specifically developed to investigate multidimensional parameter space driving photobiological interactions triggered by light (photobiomodulation) in the skin cells. The approach is based on the so-called design of experiment (DoE), a statistical method, which allows for the investigation of multidimensional parameters landscapes. This goes hand in hand with sharing practical tips for the design of light-based devices inducing these effects. To exemplify practical applications of the developed methods and light-based devices, we disclose experimental data sets and emphasize robustness and reproducibility of the results.

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.

Porphyrins and related macrocycles: Combining photosensitization with radio- or optical-imaging for next generation theranostic agents.

This review summarises recent research into combining the photosensitizing properties of porphyrins with imaging techniques such as PET and NIR fluorescence for so called "theranostic" applications, which combine biomedical imaging and therapeutic potential into a single administered substance. The photophysical mechanisms of both the therapeutic and diagnostic properties of porphyrins are discussed, as well as key characteristics that are required in order to deliver the most effective treatment.

Fluorescence from a single Symbiodinium cell.

The partnership between coral and its algal symbionts, Symbiodinium, is crucial to the global environment. Yet, the regulatory process within the photosynthetic machinery of Symbiodinium is still not clearly understood. Here, we studied the influence of light stress from focussed red and blue lasers on single Symbiodinium cells. Fluorescence signals were measured to show cell response. Increasing the incident laser power or the exposure time resulted in an increase followed by a decline in fluorescence intensity. The trend of fluorescence intensity changes was associated with mechanisms of light use efficiency, non-photochemical quenching, photoinhibition, and repair of the cell. Our study provides new approaches to studying the photobiology and physiology of Symbiodinium cells.

Calibration matters: On the procedure of using the chlorophyll fluorescence method to estimate mesophyll conductance.

Estimates of mesophyll conductance (gm), when calculated from chlorophyll fluorescence, are uncertain, especially when the photosystem II (PSII) operating efficiency is measured from the traditional single saturation pulse methodology. The multiphase flash method has recently been recommended to replace the single saturation pulse method, allowing a more reliable estimation of gm. Also, many researchers still directly use the PSII operating efficiency to derive linear electron transport rate J (that is required to estimate gm), without appropriate calibration using measurements under non-photorespiratory conditions. Here we demonstrate for tomato and rice that (i) using the multiphase flash method did not yield realistic estimates of gm if no calibration was conducted; and (ii) using the single saturation pulse method still gave reasonable estimates of gm when calibration based on the non-photorespiratory measurements was properly conducted. Therefore, conducting calibration based on data under non-photorespiratory conditions was indispensable for a reliable estimation of gm, regardless whether the multiphase flash or the single saturation pulse method was used for measuring the PSII operating efficiency. Other issues related to the procedure of using the chlorophyll fluorescence method to estimate gm were discussed.

Acclimation strategy of Rhodopseudomonas palustris to high light irradiance.

The ability of Rhodopseudomonas palustris cells to rapidly acclimate to high light irradiance is an essential issue when cells are grown under sunlight. The aim of this study was to investigate the photo-acclimation process in Rhodopseudomonas palustris 42OL under different culturing conditions: (i) anaerobic (AnG), (ii) aerobic (AG), and (iii) under H2-producing (HP) conditions both at low (LL) and high light (HL) irradiances. The results obtained clearly showed that the photosynthetic unit was significantly affected by the light irradiance at which Rp. palustris 42OL was grown. The synthesis of carotenoids was affected by both illumination and culturing conditions. At LL, lycopene was the main carotenoid synthetized under all conditions tested, while at HL under HP conditions, it resulted the predominant carotenoid. Oppositely, under AnG and AG at HL, rhodovibrin was the major carotenoid detected. The increase in light intensity produced a deeper variation in light-harvesting complexes (LHC) ratio. These findings are important for understanding the ecological distribution of PNSB in natural environments, mostly characterized by high light intensities, and for its growth outdoors.

Algal Photobiology: A Rich Source of Unusual Light Sensitive Proteins for Synthetic Biology and Optogenetics.

The light absorption system in eukaryotic (micro)algae includes highly sensitive photoreceptors, which change their conformation in response to different light qualities on a subsecond time scale and induce physiological and behavioral responses. Some of the light sensitive modules are already in use to engineer and design photoswitchable tools for control of cellular and physiological activities in living organisms with various degrees of complexity. Thus, identification of new light sensitive modules will not only extend the source material for the generation of optogenetic tools but also foster the development of new light-based strategies in cell signaling research. Apart from searching for new proteins with suitable light-sensitive modules, smaller variants of existing light-sensitive modules would be helpful to simplify the construction of hybrid genes and facilitate the generation of mutated and chimerized modules. Advances in genome and transcriptome sequencing as well as functional analysis of photoreceptors and their interaction partners will help to discover new light sensitive modules.

Controlling Protein Activity and Degradation Using Blue Light.

Regulation of protein stability is a fundamental process in eukaryotic cells and pivotal to, e.g., cell cycle progression, faithful chromosome segregation, or protein quality control. Synthetic regulation of protein stability requires conditional degradation sequences (degrons) that induce a stability switch upon a specific signal. Fusion to a selected target protein permits to influence virtually every process in a cell. Light as signal is advantageous due to its precise applicability in time, space, quality, and quantity. Light control of protein stability was achieved by fusing the LOV2 photoreceptor domain of Arabidopsis thaliana phototropin1 with a synthetic degron (cODC1) derived from the carboxy-terminal degron of ornithine decarboxylase to obtain the photosensitive degron (psd) module. The psd module can be attached to the carboxy terminus of target proteins that are localized to the cytosol or nucleus to obtain light control over their stability. Blue light induces structural changes in the LOV2 domain, which in turn lead to activation of the degron and thus proteasomal degradation of the whole fusion protein. Variants of the psd module with diverse characteristics are useful to fine-tune the stability of a selected target at permissive (darkness) and restrictive conditions (blue light).

Effect of laser optoperforation of the zona pellucida on mouse embryo development in vitro.

The effect of laser optical perforation of the zona pellucida on the viability and development of mouse embryos has been studied. Operations of zona pellucida thinning and single or double perforation were carried out on 2-cell embryo, morula, and blastocyst stages with a laser pulse (wavelength 1.48 µm, pulse duration 2 ms). Embryo development up to the blastocyst stage and hatching efficiency were statistically analyzed. It was found that 2-cell or morula stage embryo zona pellucida thinning or single perforation did not affect development to the blastocyst stage and number of hatched embryos, but it accelerated embryo hatching compared to control groups one day earlier in vitro. Double optoperforation on 2-cell embryo or morula stage did not significantly affect development to the blastocyst stage, but it strongly decreased the number of hatched embryos. Also, zona pellucida perforation at the blastocyst stage had a negative effect: hatching did not occur after this manipulation. Blastocyst cell number calculation after single zona pellucida perforation at 2-cell and morula stages showed that cell number of hatching or hatched blastocysts did not differ from the same control groups. This fact points out that the laser single optoperforation method is a useful and safe experimental tool that allows further manipulations within the zona pellucida.

Analysis of Light Transport Features in Stone Fruits Using Monte Carlo Simulation.

The propagation of light in stone fruit tissue was modeled using the Monte Carlo (MC) method. Peaches were used as the representative model of stone fruits. The effects of the fruit core and the skin on light transport features in the peaches were assessed. It is suggested that the skin, flesh and core should be separately considered with different parameters to accurately simulate light propagation in intact stone fruit. The detection efficiency was evaluated by the percentage of effective photons and the detection sensitivity of the flesh tissue. The fruit skin decreases the detection efficiency, especially in the region close to the incident point. The choices of the source-detector distance, detection angle and source intensity were discussed. Accurate MC simulations may result in better insight into light propagation in stone fruit and aid in achieving the optimal fruit quality inspection without extensive experimental measurements.

Spectral effects on Symbiodinium photobiology studied with a programmable light engine.

The spectral light field of Symbiodinium within the tissue of the coral animal host can deviate strongly from the ambient light field on a coral reef and that of artificial light sources used in lab studies on coral photobiology. Here, we used a novel approach involving light microsensor measurements and a programmable light engine to reconstruct the spectral light field that Symbiodinium is exposed to inside the coral host and the light field of a conventional halogen lamp in a comparative study of Symbiodinium photobiology. We found that extracellular gross photosynthetic O2 evolution was unchanged under different spectral illumination, while the more red-weighted halogen lamp spectrum decreased PSII electron transport rates and there was a trend towards increased light-enhanced dark respiration rates under excess irradiance. The approach provided here allows for reconstructing and comparing intra-tissue coral light fields and other complex spectral compositions of incident irradiance. This novel combination of sensor technologies provides a framework to studying the influence of macro- and microscale optics on Symbiodinium photobiology with unprecedented spectral resolution.

High-performance lighting evaluated by photobiological parameters.

The human reception of light includes image-forming and non-image-forming effects which are triggered by spectral distribution and intensity of light. Ideal lighting is similar to daylight, which could be evaluated by spectral or chromaticity match. LED-based and CFL-based lighting were analyzed here, proposed according to spectral and chromaticity match, respectively. The photobiological effects were expressed by effectiveness for blue light hazard, cirtopic activity, and photopic vision. Good spectral match provides light with more similar effects to those obtained by the chromaticity match. The new parameters are useful for better evaluation of complex human responses caused by lighting.