LOV-based fluorescent proteins (FPs) are an alternative class of fluorescent reporters with unique properties which complement the well-established proteins of the GFP family. One of the most important features of LOV-based FPs is the independence of molecular oxygen for the development of their specific fluorescence. Furthermore, they are characterized by small size and rapid signal development. Over the last few years, a number of different bacterial and plant LOV-based fluorescent proteins such as FbFP, iLOV and miniSOG have been developed and optimized. In this report, we comparatively have characterized the photophysical properties of nine different LOV-based fluorescent proteins including the excitation and emission maxima, the extinction coefficient, the fluorescence quantum yield, the average fluorescence lifetime and the photostability. The unified characterization of the LOV-based FPs provides a useful guide to apply them as in vivo tools for quantitative analyses and biological imaging.

Background: Chloride currents in peripheral nociceptive neurons have been implicated in the generation of afferent nociceptive signals, as Cl⁻ accumulation in sensory endings establishes the driving force for depolarizing, and even excitatory, Cl⁻ currents. The intracellular Cl⁻ concentration can, however, vary considerably between individual DRG neurons. This raises the question, whether the contribution of Cl⁻ currents to signal generation differs between individual afferent neurons, and whether the specific Cl⁻ levels in these neurons are subject to modulation. Based on the hypothesis that modulation of the peripheral Cl⁻ homeostasis is involved in the generation of inflammatory hyperalgesia, we examined the effects of inflammatory mediators on intracellular Cl⁻ concentrations and on the expression levels of Cl⁻ transporters in rat DRG neurons. Results: We developed an in vitro assay for testing how inflammatory mediators influence Cl⁻ concentration and the expression of Cl⁻ transporters. Intact DRGs were treated with 100 ng/ml NGF, 1.8 μM ATP, 0.9 μM bradykinin, and 1.4 μM PGE₂ for 1–3 hours. Two-photon fluorescence lifetime imaging with the Cl⁻-sensitive dye MQAE revealed an increase of the intracellular Cl⁻ concentration within 2 hours of treatment. This effect coincided with enhanced phosphorylation of the Na⁺-K⁺−2Cl⁻ cotransporter NKCC1, suggesting that an increased activity of that transporter caused the early rise of intracellular Cl⁻ levels. Immunohistochemistry of NKCC1 and KCC2, the main neuronal Cl⁻ importer and exporter, respectively, exposed an inverse regulation by the inflammatory mediators. While the NKCC1 immunosignal increased, that of KCC2 declined after 3 hours of treatment. In contrast, the mRNA levels of the two transporters did not change markedly during this time. These data demonstrate a fundamental transition in Cl⁻ homeostasis toward a state of augmented Cl⁻ accumulation, which is induced by a 1–3 hour treatment with inflammatory mediators. Conclusion: Our findings indicate that inflammatory mediators impact on Cl⁻ homeostasis in DRG neurons. Inflammatory mediators raise intracellular Cl⁻ levels and, hence, the driving force for depolarizing Cl⁻ efflux. These findings corroborate current concepts for the role of Cl⁻ regulation in the generation of inflammatory hyperalgesia and allodynia. As the intracellular Cl⁻ concentration rises in DRG neurons, afferent signals can be boosted by excitatory Cl⁻ currents in the presynaptic terminals. Moreover, excitatory Cl⁻ currents in peripheral sensory endings may also contribute to the generation or modulation of afferent signals, especially in inflamed tissue.

Background: Molecular oxygen (O₂) is one of the key metabolites of all obligate and facultative aerobic pro- and eukaryotes. It plays a fundamental role in energy homeostasis whereas oxygen deprivation, in turn, broadly affects various physiological and pathophysiological processes. Therefore, real-time monitoring of cellular oxygen levels is basically a prerequisite for the analysis of hypoxia-induced processes in living cells and tissues. Results: We developed a genetically encoded Förster resonance energy transfer (FRET)-based biosensor allowing the observation of changing molecular oxygen concentrations inside living cells. This biosensor named FluBO (fluorescent protein-based biosensor for oxygen) consists of the yellow fluorescent protein (YFP) that is sensitive towards oxygen depletion and the hypoxia-tolerant flavin-binding fluorescent protein (FbFP). Since O₂ is essential for the formation of the YFP chromophore, efficient FRET from the FbFP donor domain to the YFP acceptor domain only occurs in the presence but not in the absence of oxygen. The oxygen biosensor was used for continuous real-time monitoring of temporal changes of O₂ levels in the cytoplasm of Escherichia coli cells during batch cultivation. Conclusions: FluBO represents a unique FRET-based oxygen biosensor which allows the non-invasive ratiometric readout of cellular oxygen. Thus, FluBO can serve as a novel and powerful probe for investigating the occurrence of hypoxia and its effects on a variety of (patho)physiological processes in living cells.

Photothermal methods are currently being employed in a variety of research areas, ranging from materials science to environmental monitoring. Despite the common term which they are collected under, the implementations of these techniques are as diverse as the fields of application. In this review, we concentrate on the recent applications of time-resolved methods in photochemistry and photobiology.

Flavin-binding fluorescent proteins (FPs) are genetically encoded in vivo reporters, which are derived from microbial and plant LOV photoreceptors. In this study, we comparatively analyzed ROS formation and light-driven antimicrobial efficacy of eleven LOV-based FPs. In particular, we determined singlet oxygen (¹O₂) quantum yields and superoxide photosensitization activities via spectroscopic assays and performed cell toxicity experiments in E. coli. Besides miniSOG and SOPP, which have been engineered to generate ¹O₂, all of the other tested flavoproteins were able to produce singlet oxygen and/or hydrogen peroxide but exhibited remarkable differences in ROS selectivity and yield. Accordingly, most LOV-FPs are potent photosensitizers, which can be used for light-controlled killing of bacteria. Furthermore, the two variants Pp2FbFP and DsFbFP M49I, exhibiting preferential photosensitization of singlet oxygen or singlet oxygen and superoxide, respectively, were shown to be new tools for studying specific ROS-induced cell signaling processes. The tested LOV-FPs thus further expand the toolbox of optogenetic sensitizers usable for a broad spectrum of microbiological and biomedical applications.