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[Display omitted] • Photooxidation processes of PCDTBT polymer thin film were tuned by additives. • UV–vis absorption photobleaching degree correlates with paramagnetic defects kinetics. • 2% admixture of fluorinated carbon nanotubes inhibits photooxidation of PCDTBT. The influence of the additives to the thin film of the semiconducting polymer PCDTBT on the kinetics of its photooxidation in air was revealed through changes in the UV–vis absorption spectra and the EPR spectra of the paramagnetic defects. These defects appeared in the initial stage of the PCDTBT photooxidation, and the kinetics of their generation and decay reflected the impact of the additives on PCDTBT stability. While a small amount of PCBM and beta-carotene resulted in the accelerated photooxidation, the addition of fluorinated carbon nanotubes stabilized PCDTBT. [ABSTRACT FROM AUTHOR]

Variations in the fluorescence lifetimes of Radachlorin photosensitizers in HeLa and A549 cells, caused by photodynamic treatment, were studied using fluorescence lifetime imaging microscopy (FLIM). An analysis of FLIM images of the cells demonstrated a substantial decrease in the mean Radachlorin fluorescence lifetime and intensity as a result of UV irradiation of the photosensitized cells at different doses, with higher doses causing a more pronounced decrease in the mean fluorescence lifetime in cells. The post-treatment decrease in Radachlorin fluorescence intensity was accompanied by the appearance of an additional rapidly decaying fluorescence component and a nonlinear decrease in the weighted fluorescence lifetime obtained from double-exponential fits of time-resolved fluorescence signals. Experiments performed in the aqueous solutions of the photosensitizer revealed similar irreversible changes in the Radachlorin fluorescence lifetime and intensity. Therefore, the observed phenomena occurred most likely due to the photodegradation of the photosensitizer molecules and can be applied for dosimetry and monitoring of irradiation doses in different areas of malignant tissues in the course of photodynamic treatment.

Background: Population voltage imaging is used for studying brain physiology and brain circuits. Using a genetically encoded voltage indicator (GEVI), “VSFP” or “ASAP2s”, or a voltage-sensitive dye, Di-4-Anepps, we conducted population voltage imaging in brain slices. The resulting optical signals, optical local field potentials (LFPs), were used to evaluate the performances of the 3 voltage indicators. Methods: In brain slices prepared from VSFP-transgenic or ASAP2s-transgenic mice, we performed multi-site optical imaging of evoked cortical depolarizations - compound excitatory postsynaptic potentials (cEPSPs). Optical signal amplitudes (ΔF/F) and cEPSP decay rates (OFF rates) were compared using analysis of variance (ANOVA) followed by unpaired Student’s t test (31–104 data points per voltage indicator). Results: The ASAP2s signal amplitude (ΔF/F) was on average 3 times greater than Di-4-Anepps, and 7 times greater than VSFP. The optical cEPSP decay (OFF rate) was the slowest in Di-4-Anepps and fastest in ASAP2s. When ASAP2s expression was weak, we observed slow, label-free (autofluorescence, metabolic) optical signals mixed into the ASAP2s traces. Fast hyperpolarizations, that typically follow depolarizing cortical transients (afterhyperpolarizations), were prominent in ASAP2s but not present in the VSFP and Di-4-Anepps experiments. Conclusions: Experimental applications for ASAP2s may potentially include systems neuroscience studies that require voltage indicators with large signal amplitude (ΔF/F), fast decay times (fast response time is needed for monitoring high frequency brain oscillations), and/or detection of brain patches in transiently hyperpolarized states (afterhyperpolarization).

Bleaching adaptation in rod photoreceptors is mediated by apo-opsin, which activates phototransduction with effective activity 105- to 106-fold lower than that of photoactivated rhodopsin (meta II). However, the mechanism that produces such low opsin activity is unknown. To address this question, we sought to record single opsin responses in mouse rods. We used mutant mice lacking efficient calcium feedback to boosts rod responses and generated a small fraction of opsin by photobleaching ∼1% of rhodopsin. The bleach produced a dramatic increase in the frequency of discrete photoresponse-like events. This activity persisted for hours, was quenched by 11-cis-retinal, and was blocked by uncoupling opsin from phototransduction, all indicating opsin as its source. Opsin-driven discrete activity was also observed in rods containing non-activatable rhodopsin, ruling out transactivation of rhodopsin by opsin. We conclude that bleaching adaptation is mediated by opsin that exists in equilibrium between a predominant inactive and a rare meta II-like state.SIGNIFICANCE STATEMENT Electrophysiological analysis is used to show that the G-protein-coupled receptor opsin exists in equilibrium between a predominant inactive and a rare highly active state that mediates bleaching adaptation in photoreceptors.

The present study examines the notion that polarized exocytosis in the tips of growing hyphae creates an excess of plasma membrane and thus the need for its removal by endocytosis. To measure endocytosis experimentally, we developed a photobleaching (FRAP) procedure to count endocytic events in hyphae of Neurospora crassa carrying a fluorescent tag on the actin-binding protein fimbrin (FIM-1-GFP). Given 40 nm as the average diameter of endocytic vesicles, we calculated that about 12.5% of the plasma membrane discharged in the apex becomes endocytosed in the subapex. According to our calculations, the GFP-tagged hyphae of N. crassa, measured under the constrained conditions of confocal microscopic examination, needed about 8800 vesicles/min to extend their plasma membrane or about 9800/min, if we include predicted demands for cell wall growth and extracellular secretion. Our findings support the notion that exocytosis and endocytosis operate in tandem with the latter serving as a compensatory process to remove any excess of plasma membrane generated by the intense exocytosis in the hyphal tips. Presumably, this tandem arrangement evolved to support the hallmark features of fungi namely rapid cell extension and abundant secretion of hydrolytic enzymes.

Plant virus-based vectors are valuable tools for recombinant gene expression and functional genomics for both basic and applied research. In this study, Lettuce infectious yellows virus (LIYV) of the genus Crinivirus was engineered into a virus vector that is applicable for efficient protein expression and virus-induced gene silencing (VIGS) in plants. We examined gene replacement and “add a gene” strategies to develop LIYV-derived vectors for transient expression of the green fluorescent protein (GFP) reporter in Nicotiana benthamiana plants. The latter yielded higher GFP expression and was further examined by testing the effects of heterologous controller elements (CEs). A series of five vector constructs with progressively extended LIYV CP sgRNA CEs were tested, the longest CE gave the highest GFP expression but lower virus accumulation. The whitefly transmissibility of the optimized vector construct to other host plants, and the capability to accommodate and express a larger gene, a 1.8 kb β-glucuronidase (GUS) gene, were confirmed. Furthermore, the LIYV vector was also validated VIGS by silencing the endogenous gene, phytoene desaturase (PDS) in N. benthamiana plants, and the transgene GFP in N. benthamiana line 16c plants. Therefore, LIYV-derived vectors could provide a technical reference for developing vectors of other economically important criniviruses.

We exploit single‐molecule tracking and optical single channel recording in droplet interface bilayers to resolve the assembly pathway and pore formation of the archetypical cholesterol‐dependent cytolysin nanopore, Perfringolysin O. We follow the stoichiometry and diffusion of Perfringolysin O complexes during assembly with 60 ms temporal resolution and 20 nm spatial precision. Our results suggest individual nascent complexes can insert into the lipid membrane where they continue active assembly. Overall, these data support a model of stepwise irreversible assembly dominated by monomer addition, but with infrequent assembly from larger partial complexes. [ABSTRACT FROM AUTHOR]

Stable subphthalocyanine-type dyes with a high electron affinity attract much attention as potential substitutes for traditional fullerenes in molecular electronics devices. One possible way to enhance the acceptor properties of the subphthalocyanine core is by replacing the peripheral benzene fragments (C6H4) with 1,2,5-thiadiazole fragments (C2N2S1). However, the resistance of these materials to light or atmospheric effect remains an open question, which limits their further application in device manufacturing. In this work, we compare vacuum-deposited films of three derivatives, SubPzS3H0 (all peripheral fragments are 1,2,5-thiadiazoles), SubPzS2H4 (two fragments are 1,2,5-thiadiazoles and one fragment is benzene), and SubPcS0H12 (all benzenes, i.e., parent subphthalocyanine). Practically relevant substrates were used for deposition, namely, bare glass, glass/ITO or FTO, and PET/ITO. Photobleaching of films under continuous 1 sun illumination was studied in laboratory air, synthetic air, and ultrapure argon. It is shown that the exclusion of near-UV photons from the incident light spectrum, which corresponds to the absorption of subphthalocyanines in the Soret-band, strongly inhibits degradation. Absorption in the Q-band range initiates soft annealing rather than photobleaching of films. The stability of the films deposited on glass decreases as SubPzS3H0 > SubPzS2H4 > SubPcS0H12 in air, and vice versa in argon. The substrate adds more complexity to this picture. In argon, the ITO coating reduces degradation of all of the compounds equally, in contrast to the glass samples, while in air, the SubPzS3H0 films discolor completely. The latter reaction proceeds due to the indium-containing species migrating from the conductive coating. [ABSTRACT FROM AUTHOR]

Organometallic photosensitizers are very efficient triplet sensitizers that have found wide use in photocatalysis, but they are scarce, toxic, and expensive. Organic fluorophores have also been used in several systems, but they can suffer from lower efficiency and sensitivity to photobleaching. In this work, we immobilized ruthenium (II) tris(bipyridine) and rose bengal on magnetic nanosupports and compared their activity and photostability in the singlet oxygen mediated photooxidation of citronellol. Both systems can perform the transformation with equivalent turnover frequencies and be magnetically recovered. We studied their long-term stability and found photobleaching to be a major cause of photocatalyst deactivation over time for both systems, and we were able to improve their photostability upon immobilization on solid supports. [Display omitted] • Fe@SiO 2 NPs can be used as magnetic supports to afford recyclable photocatalysts for singlet oxygen mediated oxidation. • Photobleaching is an important cause of catalyst deactivation for both rose bengal and ruthenium (II) tris(bipyridine). • Immobilization improved the photostability of photosensitizers rose bengal and ruthenium (II) tris(bipyridine). [ABSTRACT FROM AUTHOR]

Bright and stable emitters of single indistinguishable photons are crucial for quantum technologies. The origin of the promising bright emitters recently observed in hexagonal boron nitride (hBN) still remains unclear. This study reports pure single-photon sources in multi-layered hBN at room temperature that demonstrate high emission rates. The quantum emitters are introduced with argon beam treatment and air annealing of mechanically exfoliated hBN flakes with thicknesses of 5–100 nm. Spectral and time-resolved measurements reveal the emitters have more than 1 GHz of excited-to-ground state transition rate. The observed photoswitching between dark and bright states indicates the strong sensitivity of the emitter to the electrostatic environment and the importance of the indirect excitation for the photodynamics. [ABSTRACT FROM AUTHOR]

The ORBEYE (ORB), an innovative 3-dimensional digital exoscope, is an equipped system for fluorescence-guided surgery with 5-aminolevulinic acid. Therefore, this study aimed to verify the characteristics of fluorescence-guided surgery with 5-aminolevulinic acid and excitation light source with ORB. The same operative field of glioblastoma was recorded under blue light (BL) excitation using a conventional microscope (MS) and ORB. For in vitro studies, the energy of 405-nm wavelength light in white light and BL modes of each scope was examined in various focal lengths. To examine the degree of photobleaching with BL for each scope, protoporphyrin IX–soaked filter papers were continuously exposed with BL of an MS and ORB, and the video-recorded red fluorescence intensity was analyzed. The color tone of tumor-induced red fluorescence was remarkably different under each scope. Furthermore, nonfluorescent normal structures without red fluorescence were well recognized under ORB. The energy of 405-nm wavelength light in BL was significantly higher in ORB than that in an MS, especially in the short focal length. With continuous BL excitation to filter papers, the relative red fluorescence intensity of filter papers was significantly decreased over time in ORB than in an MS. In low protoporphyrin IX concentration, the difference was more significant. With ORB, the good visibility due to BL energy as compared with an MS might improve the surgical manipulation even in BL mode. However, the weak fluorescent tissue and short focal length should be carefully considered because photobleaching might be critical for FGS. [ABSTRACT FROM AUTHOR]

Biomolecular systems, such as proteins, crucially rely on dynamic processes at the nanoscale. Detecting biomolecular nanodynamics is therefore key to obtaining a mechanistic understanding of the energies and molecular driving forces that control biomolecular systems. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to observe in real-time how a single biomolecule proceeds through its functional cycle involving a sequence of distinct structural states. Currently, this technique is fundamentally limited by irreversible photobleaching, causing the untimely end of the experiment and thus, a narrow temporal bandwidth of ≤ 3 orders of magnitude. Here, we introduce "DyeCycling", a measurement scheme with which we aim to break the photobleaching limit in smFRET. We introduce the concept of spontaneous dye replacement by simulations, and as an experimental proof-of-concept, we demonstrate the intermittent observation of a single biomolecule for one hour with a time resolution of milliseconds. Theoretically, DyeCycling can provide > 100-fold more information per single molecule than conventional smFRET. We discuss the experimental implementation of DyeCycling, its current and fundamental limitations, and specific biological use cases. Given its general simplicity and versatility, DyeCycling has the potential to revolutionize the field of time-resolved smFRET, where it may serve to unravel a wealth of biomolecular dynamics by bridging from milliseconds to the hour range. [ABSTRACT FROM AUTHOR]

Biomass burning is an important source of brown carbon (BrC) aerosols, which influence climate by affecting the Earth's radiative balance. However, the transformation pathways of BrC chromophores, especially in the presence of photochemically active species, such as nitrate, are not well understood. In this study, the nitrate-mediated aqueous-phase photooxidation of three typical BrC chromophores from biomass burning was investigated, including 4-nitrocatechol, 3-nitrosalicylic acid, and 3,4-dinitrophenol. Variations in nitrate concentrations, pH, and temperatures were systematically examined to assess their impacts on the apparent photolysis rates of these BrC chromophores. The results show that increasing nitrate concentrations significantly enhances apparent photolysis rates to 3-3.5 times compared to nitrate-free conditions. Also, a temperature rise from 0 to 30 °C increases apparent photolysis rates by a factor of 1.3-2.5 for these chromophores. However, the effect of pH varies among these chromophores, depending on the substituents and their positions on the benzene ring. High-resolution mass spectrometric analysis suggests that the photooxidation of these chromophores initiates with the addition of nitro and/or hydroxyl groups to the benzene ring, followed by a ring-opening reaction and the formation of smaller, highly oxygenated molecules including formic acid, glyoxylic acid, malonic acid, and nitropropanoic acid. This study highlights the key role of nitrate in the aqueous-phase photooxidation of BrC, altering the aging pathways and shortening the atmospheric lifetimes of BrC. These results are of particular importance for a better understanding of BrC aging and its radiative forcing, given the increase of the nitrate mass fraction in aerosols of China in recent years.

Camera-based single-molecule techniques have emerged as crucial tools in revolutionizing the understanding of biochemical and cellular processes due to their ability to capture dynamic processes with high precision, high-throughput capabilities, and methodological maturity. However, the stringent requirement in photon number per frame and the limited number of photons emitted by each fluorophore before photobleaching pose a challenge to achieving both high temporal resolution and long observation times. In this work, we introduce MUFFLE, a supervised deep-learning denoising method that enables single-molecule FRET with up to 10-fold reduction in photon requirement per frame. In practice, MUFFLE extends the total number of observation frames by a factor of 10 or more, greatly relieving the trade-off between temporal resolution and observation length and allowing for long-term measurements even without the need for oxygen scavenging systems and triplet state quenchers., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Single-molecule photobleaching analysis is a useful approach for quantifying reactive membrane protein oligomerization in membranes. It provides a binary readout of a fluorophore attached to a protein subunit at dilute conditions. However, quantification of protein stoichiometry from this data requires information about the subunit labeling yields and whether there is non-specific background labeling. Any increases in subunit-specific labeling improves the ability to determine oligomeric states with confidence. A common strategy for site-specific labeling is by conjugation of a fluorophore bearing a thiol-reactive maleimide group to a substituted cysteine. Yet, cysteine reactivity can be difficult to predict as it depends on many factors such as solvent accessibility and electrostatics from the surrounding protein structure. Here we report a general methodology for screening potential cysteine labeling sites on purified membrane proteins. We present the results of two example systems for which the dimerization reactions in membranes have been characterized: (1) the CLC-ec1 Cl - /H + antiporter, an Escherichia coli homologue of voltage-gated chloride ion channels in humans and (2) a mutant form of a member of the family of fluoride channels Fluc from Bordetella pertussis (Fluc-Bpe-N43S). To demonstrate how we identify such sites, we first discuss considerations of residue positions hypothesized to be suitable and then describe the specific steps to rigorously assess site-specific labeling while maintaining functional activity and robust single-molecule fluorescence signals. We find that our initial, well rationalized choices are not strong predictors of success, as rigorous testing of the labeling sites shows that only ≈ 30 % of sites end up being useful for single-molecule photobleaching studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Correlative super-resolution microscopy has the potential to accurately visualize and validate new biological structures past the diffraction limit. However, combining different super-resolution modalities, such as deterministic stimulated emission depletion (STED) and stochastic single-molecule localization microscopy (SMLM), is a challenging endeavour. For correlative STED and SMLM, the following poses a significant challenge: (1) the photobleaching of the fluorophores in STED; (2) the subsequent reactivation of the fluorophores for SMLM and (3) finding the right fluorochrome and imaging buffer for both imaging modalities. Here, we highlight how the deep ultraviolet (DBUE) wavelengths of the Mercury (Hg) arc lamp can help recover STED bleaching and allow for the reactivation of single molecules for SMLM imaging. We also show that Alexa Fluor 594 and the commercially available Prolong Diamond to be excellent fluorophores and imaging media for correlative STED and SMLM., (© 2023 The Author(s). Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.)

In modern times, patients are not only seeking oral health but also aiming for a flawless smile. Despite the progress made in aesthetic dentistry, there are instances where insufficient teeth whitening results contrast with the considerable cost of professional treatments. Patient discomfort and the potential for tooth damage further compound these challenges. In this study, we conducted a comparative split-mouth ex vivo investigation to evaluate the innovative BlancOne ULTRA+ (IDS SpA) in comparison with established professional products: Opalescence Extraboost (ULTRADENT), Zoom WhiteSpeed (Philips Research Eindhoven High Tech), and Pola Office (SDI Limited). Our initial focus was on the whitening effectiveness of each product, which was measured using a spectrophotometer. Subsequently, we assessed any structural changes in enamel post-treatment using an atomic force microscope (AFM) and a nanoindentation procedure. All tested bleaching agents demonstrated teeth-brightening effects. BlancOne ULTRA+, Zoom WhiteSpeed, and Pola Office caused minor alterations in the texture of the enamel surface within nanometric limits. However, Opalescence Extraboost exhibited notably more pronounced changes, indicating significant modifications in surface roughness and potential reductions in material hardness due to consequential shifts in mechanical properties. BlancOne ULTRA+ appears to offer the most favourable cost–benefit outcome.

Carbon dots (CDs) are novel fluorescent nanoparticles that combine intense emission of visible light with eco-friendly and inexpensive carbon-based composition. In this work, CDs are synthesized trough a glycothermal treatment of resorcinol (1,3-hydroxybenzene) in air atmosphere. The presence of catalysts (NaOH and H 2 SO 4) increases the reaction rate, promoting a faster and massive production of nanoparticles. The spectroscopic monitoring of fluorescence during CD synthesis, supported by a DFT study, allows to depict the formation and structural evolution of OH terminated polycyclic aromatic hydrocarbons (PAHs) from resorcinol polycondensation. In purified CDs, PAHs embedded in the amorphous carbogenic core are responsible for an intense green fluorescence emission with a quantum yield up to ∼40%. Such band exhibits high resistance to UV photobleaching, attributed to the physical protection of the carbogenic matrix. Finally, adding a strong acid/base to the CD solution, the CD fluorescence can be cyclically quenched/restored (due to reversible aggregation), suggesting the convenient use of such CDs in on/off sensors or stimulus-responding devices. [Display omitted] • Carbon Dots (CDs) synthesized via resorcinol carbonization in open reaction vessel. • Strong base and acid catalysts improve the CDs reaction yield. • Intense green fluorescence ascribed to PAHs embedded in amorphous carbogenic core. • UV photobleaching resistance due to physical protection of PAHs from the CD matrix. • Cyclical on-off fluorescence by addition of strong base/acid to CD solution. [ABSTRACT FROM AUTHOR]

Background: Image segmentation in fluorescence microscopy is often based on spectral separation of fluorescent probes (color-based segmentation) or on significant intensity differences in individual image regions (intensity-based segmentation). These approaches fail, if dye fluorescence shows large spectral overlap with other employed probes or with strong cellular autofluorescence. Results: Here, a novel model-free approach is presented which determines bleaching characteristics based on dynamic mode decomposition (DMD) and uses the inferred photobleaching kinetics to distinguish different probes or dye molecules from autofluorescence. DMD is a data-driven computational method for detecting and quantifying dynamic events in complex spatiotemporal data. Here, DMD is first used on synthetic image data and thereafter used to determine photobleaching characteristics of a fluorescent sterol probe, dehydroergosterol (DHE), compared to that of cellular autofluorescence in the nematode Caenorhabditis elegans. It is shown that decomposition of those dynamic modes allows for separating probe from autofluorescence without invoking a particular model for the bleaching process. In a second application, DMD of dye-specific photobleaching is used to separate two green-fluorescent dyes, an NBD-tagged sphingolipid and Alexa488-transferrin, thereby assigning them to different cellular compartments. Conclusions: Data-based decomposition of dynamic modes can be employed to analyze spatially varying photobleaching of fluorescent probes in cells and tissues for spatial and temporal image segmentation, discrimination of probe from autofluorescence and image denoising. The new method should find wide application in analysis of dynamic fluorescence imaging data. [ABSTRACT FROM AUTHOR]

• Colored dissolved organic matter (CDOM) is being received and degraded in lakes. • Simultaneous exposure to irradiation and microbial activity promoted high CDOM degradation. • CDOM absorption (340 nm) declined by 41% after 37 days at summer irradiation-intensities. • Time course of CDOM in a humic lake could be predicted from input and degradation. • Humic lakes retain their color due to ongoing CDOM input. Throughout the freshwater continuum, Dissolved Organic Carbon (DOC) and the colored fraction, Chromophoric Dissolved Organic Material (CDOM), are continuously being added, removed, and transformed, resulting in changes in the chromophoricity and lability of organic matter over time. We examined, experimentally, the effect of increasing irradiation-intensities on the combined photochemical and microbial degradation of CDOM and DOC. This was done by using a simulated mixed water column: aged water from a humic lake was exposed to four irradiation-intensities – representing winter, early and late spring, and summer conditions (0.10, 0.16, 0.36, and 0.58 W/m2) – and compared with dark controls over 37 days. We found a linear relationship between CDOM degradation and irradiation-intensities up to 0.36 W/m2; the degradation rate saturated at higher intensities, both at specific wavelengths and for broader intervals. After 37 days at high irradiation-intensity, CDOM absorption of irradiation at 340 nm had been reduced by 41%; 48% of DOC had been removed and DOC degradation continued to increase. Aromaticity (SUVA 254) declined significantly over 37 days at the two lowest but not at the two highest UV- intensities; levels in unexposed control water remained constant. Direct observations of the humic lake showed that CDOM absorption of irradiation (340 nm) declined by 27% from winter to summer. A model based on hydrological CDOM input and CDOM degradation calculated from field measurements of UV-radiation and experimental CDOM degradation with UV-exposure from sunlight accurately predicted the annual course as observed in the lake. With no external CDOM input, 92% of the CDOM could be degraded in a year. The results support the notion that combined photochemical and microbial CDOM degradation can be remarkably higher in lakes than previously thought and that humic lakes retain their color due to light absorption by ongoing CDOM input. [Display omitted] [ABSTRACT FROM AUTHOR]

The aging and photobleaching effects on the acid Fuchsin dye with different concentrations in the absence and the presence of poly(vinyl alcohol) were investigated. For studying the aging effect of the samples, the optical absorption properties were characterized by UV–Vis spectroscopy. Also, the photoluminescence spectrum of the samples was measured for considering the photobleaching effect. The absorbance of dye in the proximity of PVA is enhanced. Existence of PVA introducing a quenching effect in the photoluminescence intensity of dye with an increase in dye concentration. Results show that the aging value of AF dye in the presence of PVA is enhanced with an accompanying quenching of the photobleaching value. [ABSTRACT FROM AUTHOR]

The growing interest in using photodynamic therapy for cancer treatment and antimicrobial applications has prompted the search for different classes of dyes. In general, the protocols of these studies are different, making it difficult to compare their efficiency directly. Here, we apply a controlled protocol to analyze the photophysical properties of Erythrosin B, Eosin Y, and Rose Bengal using a set of optical techniques. The results show that Erythrosine has the best singlet oxygen generation capacity. This result, added to the well-known low toxicity of Erythrosine, makes it a good choice among the xanthenes for health applications. [Display omitted] • Photo-induced physicochemical characterization of xanthene dyes. • Laser-induced lens method for measuring dyes' photoactive properties. • Chemical efficiency of singlet oxygen generation in xanthene dyes. • Potential applications of Erythrosine B for PDT and PDI. [ABSTRACT FROM AUTHOR]

[Display omitted] • A virus-induced gene silencing system was successfully constructed for the first time in walnut (Juglans regia L.) seedlings. • Spray infiltration yielded a whole plant photobleaching phenotype. • JrPOR gene silencing reduced chlorophyll content. Walnut (Juglans regia L.) is a high-value tree species planted worldwide, but the incomplete less developed genetic transformation system limits its gene function analysis. In this study, virus-induced gene silencing (VIGS) mediated by tobacco rattle virus (TRV) technology was applied to walnut seedlings to degrade the transcript of target gene. Different infiltration methods were used to explore the effects of infection mode, Agrobacterium cell density, silencing fragment length, and walnut cultivars. The results showed that spray infiltration of seedlings resulted in a photobleaching phenotype of the whole plant. Leaf injection was a more effective way of infiltration. The optimal combination was the Agrobacterium cell density at OD 600 = 1.1 with target fragment = 255 bp for the treatment of walnut early-fruiting cultivar 'Xiangling.' This combination can reach up to 48 % of gene silencing efficiency. Based on this optimized VIGS system, silencing a walnut chlorophyll synthesis-related gene, JrPOR (Protochlorophyllide reductase), to further validate the system's effect. The results showed that the expression of JrPOR was significantly repressed, and the chlorophyll level of the silenced plants was significantly decreased compared with the control. The above results indicate that the walnut TRV-VIGS system has been successfully established and can be used for reverse genetic studies, providing an option for verifying gene function in walnut. [ABSTRACT FROM AUTHOR]

Abstract Background Maize dwarf mosaic virus (MDMV), a member of the genus Potyvirus, infects maize and is non-persistently transmitted by aphids. Several plant viruses have been developed as tools for gene expression and gene silencing in plants. The capacity of MDMV for both gene expression and gene silencing were examined. Results Infectious clones of an Ohio isolate of MDMV, MDMV OH5, were obtained, and engineered for gene expression only, and for simultaneous marker gene expression and virus-induced gene silencing (VIGS) of three endogenous maize target genes. Single gene expression in single insertion constructs and simultaneous expression of green fluorescent protein (GFP) and silencing of three maize genes in a double insertion construct was demonstrated. Constructs with GFP inserted in the N-terminus of HCPro were more stable than those with insertion at the N-terminus of CP in our study. Unexpectedly, the construct with two insertion sites also retained insertions at a higher rate than single-insertion constructs. Engineered MDMV expression and VIGS constructs were transmissible by aphids (Rhopalosiphum padi). Conclusions These results demonstrate that MDMV-based vector can be used as a tool for simultaneous gene expression and multi-gene silencing in maize.

Magnesium(II) porphyrazine bearing bulky sulfanyl substituents was evaluated as a photosensitizer (PS) for photodynamic antimicrobial chemotherapy (PACT). In the photostability study, the studied porphyrazine under light irradiation decomposed via photobleaching process with quantum yields up to 1.05 ⋅ 10−5. Singlet oxygen quantum yields for the PS were determined by indirect method with chemical singlet oxygen trap (1,3-diphenylisobenzofuran, DPBF) and reference (unsubstituted zinc(II) phthalocyanine). Liposomes containing porphyrazine derivative were obtained with thin-film hydration method and their size was measured with NanoSight LM10 (Malvern). Biological activity evaluation was performed with the agar plate method. The photocytotoxicity of sulfanyl porphyrazine against various wound bacteria (Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, and Streptococcus pyogenes) was studied. The highest reduction value in bacterial growth exceeding 4 log was observed for S. epidermidis. Equally high reduction values of 3.4 log and 4.2 log at 10 and 100 µM, respectively, were observed against S. pyogenes. The novel sulfanyl porphyrazine can be considered as a potential PS in further antimicrobial photodynamic therapy related studies. [ABSTRACT FROM AUTHOR]

The phase separation and aggregation of proteins are hallmarks of many neurodegenerative diseases. These processes can be studied in living cells using fluorescent protein constructs and quantitative live-cell imaging techniques, such as fluorescence recovery after photobleaching (FRAP) or the related fluorescence loss in photobleaching (FLIP). While the acquisition of FLIP images is straightforward on most commercial confocal microscope systems, the analysis and computational modeling of such data is challenging. Here, a novel model-free method is presented, which resolves complex spatiotemporal fluorescence-loss kinetics based on dynamic-mode decomposition (DMD) of FLIP live-cell image sequences. It is shown that the DMD of synthetic and experimental FLIP image series (DMD-FLIP) allows for the unequivocal discrimination of subcellular compartments, such as nuclei, cytoplasm, and protein condensates based on their differing transport and therefore fluorescence loss kinetics. By decomposing fluorescence-loss kinetics into distinct dynamic modes, DMD-FLIP will enable researchers to study protein dynamics at each time scale individually. Furthermore, it is shown that DMD-FLIP is very efficient in denoising confocal time series data. Thus, DMD-FLIP is an easy-to-use method for the model-free detection of barriers to protein diffusion, of phase-separated protein assemblies, and of insoluble protein aggregates. It should, therefore, find wide application in the analysis of protein transport and aggregation, in particular in relation to neurodegenerative diseases and the formation of protein condensates in living cells. [ABSTRACT FROM AUTHOR]

Photopolymers are an attractive option for large-format additive manufacturing (LFAM), because they can be formulated from structural thermosets and cure rapidly in ambient conditions under low-energy ultraviolet light-emitting diode (UV LED) lamps. Photopolymer cure is strongly influenced by the depth penetration of UV light, which can be limited in the 2–4 mm layer thicknesses typical of LFAM. Photoinitiator (PI) systems that exhibit photobleaching have proven useful in thick-section cure applications, because they generate a photoinitiation wavefront, but this effect is time-dependent. This study investigates the light transmission and through-thickness cure behavior in (meth)acrylate photopolymer formulations with the photobleaching initiator bis(2,4,6-trimethylbenzoyl)-phenylphosphine oxide (BAPO). Utilizing an optical model developed by Kenning et al., lower concentrations (0.1 wt% to 0.5 wt%) of BAPO were predicted to yield rapid onset of photoinitiation. In situ cure measurements under continuous UV LED irradiation of 380 mW/cm2 showed that a 0.1 wt% concentration of BAPO achieved peak polymerization rate within 2.5 s at a 3-mm depth. With only 1 s of irradiation at 1.7 W/cm2 intensity, the 0.1 wt% BAPO formulation also achieved the highest level of cure of the formulas tested. For an irradiation dose of 5.5 J/cm2 at a duration of 3.7 s, cured polymer specimens achieved a flexural strength of 108 MPa and a flexural modulus of 3.1 GPa. This study demonstrates the utility of optical modeling as a potential screening tool for new photopolymer formulations, primarily in identifying an upper limit to PI concentration for the desired cure depth. The results also show that photobleaching provides only a limited benefit for LFAM applications with short (1.0 s to 3.7 s) UV irradiation times and indicate that excess PI concentration can inhibit light transmission even under extended irradiation times up to 60 s. [ABSTRACT FROM AUTHOR]

The water-soluble chlorophyll-proteins (WSCP) of class II from Brassicaceae are non-photosynthetic proteins that bind chlorophylls (Chls) and chlorophyll derivatives. Their physiological roles, biochemical functions and mode of action are still unclear. It is assumed that the WSCPs have a protection function against Chl photodamage during stressful conditions. WSCPs are subdivided into class IIA and class IIB according to their apparent Chla/b binding ratio. Although their Chla/Chlb binding selectivity has been partly characterized, their Chl affinities are not yet precisely defined. For instance, WSCPs IIA do not show any Chl binding preference while WSCPs IIB have greater affinity to Chlb. In this study, we present a novel method for assessment of Chl binding to WSCPs based on the differences of Chl photobleaching rates in a large range of Chl/protein ratios. The protein we have chosen to study WSCP is BnD22, a WSCP IIA induced in the leaves of Brassica napus under water deficit. BnD22 formed oligomeric complexes upon binding to Chla and/or Chlb allowing a protective effect against photodamage. The binding constants indicate that BnD22 binds with high affinity the Chls and with a strong selectivity to Chla. Moreover, dependending of Chl/protein ratio upon reconstitution, two distinct binding events were detected resulting from difference of Chl stoichiometry inside oligomeric complexes. • Photobleaching is a sensitive method to measure the Chl binding affinities of WSCPs. • BnD22, a WSCP IIA, shows stronger Chl affinity and selectivity for Chla than Chlb. • BnD22 presents different affinities for Chls according to Chl/protein ratio. • Two forms of oligomeric complexes lead to biphasic process of BnD22-Chls binding. • Level of Chl photoprotection is according to the oligomeric complexes forms. [ABSTRACT FROM AUTHOR]

Amorphous chalcogenide glasses are intrinsically metastable, highly photosensitive, and therefore exhibit numerous light-induced effects upon bandgap and sub-bandgap illumination. Depending on the pulse duration of the excitation laser, ChGs exhibit a series of light-induced effects spanning over femtosecond to seconds time domain. For continuous wave (CW) illumination, the effects are dominantly metastable in terms of photodarkening (PD) and photobleaching (PB) that take place via homopolar to heteropolar bond conversion. On the other hand, under nanosecond and ultrafast pulsed illumination, ChGs exhibit transient absorption (TA) that is instigated from the transient bonding rearrangements through self-trapped exciton recombination. In the first part of the review, we pay special attention to continuous wave light-induced PD and PB, while in the second part we will focus on the TA and controlling such effects via internal and external parameters, e.g., chemical composition, temperature, sample history, etc.

Förster resonance energy transfer (FRET) is a widely used technique for nanoscale molecular distance measurements, which makes FRET ideal for studying protein interactions and quaternary structure of protein complexes. In this work, we were interested in how photobleaching of donor and acceptor molecules affects the FRET results under various excitation conditions. We conducted a systematic study, under two-photon excitation, of the effects of the excitation power and the choice of excitation wavelengths upon the measured FRET efficiencies of multiplex protein constructs, consisting of one donor (D) and two acceptors (A) or one acceptor and a non-fluorescent tag (N), using both the kinetic theory of FRET and numerical simulations under given excitation conditions. We found that under low excitation power and properly chosen excitation wavelengths the relationship between the FRET efficiency of a trimeric construct ADA agrees within 2% with the FRET efficiency computed (via the kinetic theory of FRET in the absence of photobleaching) from two dimeric constructs ADN and NDA. By contrast, at higher excitation powers the FRET efficiencies changed significantly due to the photobleaching of both the donor (through direct excitation) and the acceptor (mostly through FRET-induced excitation). Based on these results and numerical simulations using a simple but competent algorithm, we developed guidelines for choosing appropriate experimental conditions for reliable FRET measurements, as well as for interpreting the results of existing experiments using different excitation schemes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Photobleaching of fluorescent proteins (FPs) is a major limitation to their use in advanced microscopy, and improving photostability remains highly challenging due to limited understanding of its molecular mechanism. Here we discovered a new mechanism to increase FP photostability. Cysteine oxidation, implicated in only photobleaching before, was found to drastically enhance FP photostability to the contrary. We generated a far-red FP mStable by introducing a cysteine proximal to the chromophore. Upon illumination, this cysteine was oxidized to sulfinic and sulfonic acids, enabling mStable more photostable than its ancestor mKate2 by 12-fold and surpassing other far-red FPs. mStable outperformed in laser scanning confocal imaging and super-resolution structured illumination microscopy. Moreover, photosensitization to oxidize a cysteine similarly introduced in another FP mPlum also increased its photostability by 23-fold. This postfolding cysteine sulfoxidation cannot be simply substituted by the isosteric aspartic acid, representing a unique mechanism valuable for engineering better photostability into FPs.

Fluorescent proteins (FPs) are powerful tools for cell and molecular biology. Here based on structural analysis, a blue-shifted mutant of a recently engineered monomeric infrared fluorescent protein (mIFP) has been rationally designed. This variant, named iBlueberry, bears a single mutation that shifts both excitation and emission spectra by approximately 40 nm. Furthermore, iBlueberry is four times more photostable than mIFP, rendering it more advantageous for imaging protein dynamics. By tagging iBlueberry to centrin, it has been demonstrated that the fusion protein labels the centrosome in the developing zebrafish embryo. Together with GFP-labeled nucleus and tdTomato-labeled plasma membrane, time-lapse imaging to visualize the dynamics of centrosomes in radial glia neural progenitors in the intact zebrafish brain has been demonstrated. It is further shown that iBlueberry can be used together with mIFP in two-color protein labeling in living cells and in two-color tumor labeling in mice.

A series of free base meso-tetraarylporphyrins functionalized with substituents containing one, two, and four cyclooctatetraene (COT) moieties have been obtained and characterized by spectral and photophysical studies. Three COT-free porphyrins served as reference compounds. COT is a triplet quencher, well-known to enhance the photostability of several, but not all, fluorophores. In the case of porphyrins, substitution with COT improves photostability in zinc derivatives, but for free bases, the effect is the opposite. We show that placing the COT moiety further from the free base porphyrin core enhances the photostability when the COT group lies in the direct vicinity of the macrocycle. The quantum yields of photobleaching inversely correlate with porphyrin oxidation potentials. An improvement in photostability in both COT-containing and COT-free porphyrins can be achieved by screening the porphyrin core from oxygen by switching from tolyl to mesityl substituents. This leads to a decrease in the photobleaching quantum yield, even though triplet lifetimes are longer. The results confirm the involvement of oxygen in the photodegradation of porphyrins.

Photobleaching is the permanent loss of fluorescence after extended exposure to light and is a major limiting factor in super‐resolution microscopy (SRM) that restricts spatiotemporal resolution and observation time. Strategies for preventing or overcoming photobleaching in SRM are reviewed developing new probes and chemical environments. Photostabilization strategies are introduced first, which are borrowed from conventional fluorescence microscopy, that are employed in SRM. SRM‐specific strategies are then highlighted that exploit the on–off transitions of fluorescence, which is the key mechanism for achieving super‐resolution, which are becoming new routes to address photobleaching in SRM. Off states can serve as a shelter from excitation by light or an exit to release a damaged probe and replace it with a fresh one. Such efforts in overcoming the photobleaching limits are anticipated to enhance resolution to molecular scales and to extend the observation time to physiological lifespans. [ABSTRACT FROM AUTHOR]

Photobleaching is an irreversible photochemical process that makes fluorophores not to emit permanently, limiting the observation time of the molecules. Thus, preliminary photobleaching can interfere with most fluorescence spectroscopy and microscopy, especially single‐molecule fluorescence applications. To minimize photobleaching of fluorophores, low excitation power as well as specialized imaging buffers that improve the photostability of fluorophores is highly recommended in single‐molecule fluorescence measurements. Here, we investigated how photobleaching can get affected by the excitation power and buffer components in the single‐molecule buffer system such as Trolox and glucose oxidase/catalase (GOC). We found that the photobleaching rate showed higher‐order photon interaction, indicating that photobleaching rate is decoupled to the amount of fluorescence. We also observed that the photobleaching rate was significantly reduced by fourfold when the combination of Trolox and oxygen scavenging system using either GOC or N2 bubbling was employed in single‐molecule buffer. [ABSTRACT FROM AUTHOR]

Dye-encoded bead-based assays are widely used for diagnostics. Multiple bead populations are required for multiplexing and can be produced using different dye colors, labeling levels, or combinations of dye ratios. Ready-to-use multiplex bead populations restrict users to specific targets, are costly, or require specialized instrumentation. In-house methods produce few bead plexes or require many fine-tuning steps. To expand bead encoding strategies, we present a simple, safe, and cost-effective bench-top system for generating bead populations using photobleaching. By photobleaching commercially available dye-encoded magnetic beads for different durations, we produce three times as many differentiable bead populations on flow cytometry from a single dye color. Our photobleaching system uses a high-power LED module connected to a light concentrator and a heat sink. The beads are photobleached in solution homogeneously by constant mixing. We demonstrate this photobleaching method can be utilized for cross-testing antibodies, which is the first step in developing immunoassays. The assay uses multiple photobleached encoded beads conjugated with capture antibodies to test many binding pairs simultaneously. To further expand the number of antibodies that can be tested at once, several antibodies were conjugated to the same bead, forming a pooled assay. Our assay predicts the performance of antibody pairs used in ultrasensitive Simoa assays, narrowing the number of cross-tested pairs that need to be tested by at least two-thirds and, therefore, providing a rapid alternative for an initial antibody pair screening. The photobleaching system can be utilized for other applications, such as multiplexing, and for photobleaching other particles in solution.

Motor-driven cytoskeletal remodeling in cellular systems can often be accompanied by a diffusive-like effect at local scales, but distinguishing the contributions of the ordering process, such as active contraction of a network, from this active diffusion is difficult to achieve. Using light-dimerizable kinesin motors to spatially control the formation and contraction of a microtubule network, we deliberately photobleach a grid pattern onto the filament network serving as a transient and dynamic coordinate system to observe the deformation and translation of the remaining fluorescent squares of microtubules. We find that the network contracts at a rate set by motor speed but is accompanied by a diffusive-like spread throughout the bulk of the contracting network with effective diffusion constant two orders of magnitude lower than that for a freely-diffusing microtubule. We further find that on micron scales, the diffusive timescale is only a factor of ≈ 3 slower than that of advection regardless of conditions, showing that the global contraction and long-time relaxation from this diffusive behavior are both motor-driven but exhibit local competition within the network bulk., Competing Interests: The authors declare no conflict of interest.

Indocyanine green (ICG) is an FDA-approved near infrared (NIR) imaging agent for diagnosis and imaging guided surgery. It also exhibits phototoxicity under high-dose NIR irradiation, expanding its application as a photo-therapeutic agent. Since ICG's efficiency as a type II photosensitizer has been controversial due to its low triplet state yield, other mechanisms have been explored. While claims of toxic decomposition products, accompanied by irreversible ICG photobleaching, were proposed as the main mechanism, evidences from systemic studies are lacking. In this work, we aimed to unravel the factors affecting ICG photobleaching and the associated photo-killing effect on neuroblastoma, one of the most common pediatric tumors but often escapes therapy. Specifically, we examined how albumin-induced ICG stabilization affects the ICG photobleaching process, and the effect of photobleached ICG on cell proliferation and viability of neuroblastoma cells. It was found that ICG photobleaching was significant only under aerobic conditions and was more efficient in solutions with higher concentration ICG monomers, which were stabilized from aggregates by the presence of BSA while increasing photobleaching and associated oxygen consumption. Photobleached ICG inhibited cell proliferation, indicating another effect of tumor treatment by ICG. Taken together, while enhanced photobleaching by BSA-bound ICG monomers may reduce the photodynamic effect targeting cellular components, the photoproducts directly contribute to tumor growth inhibition and assist in a secondary mechanism to stop tumor growth. • Photobleached ICG inhibited neuroblastoma cell proliferation. • ICG requires and consumes dissolved oxygen for photobleaching in cells. • BSA enhances photobleaching by preventing monomers from aggregating. • Monomers are more photoreactive than aggregates. • Lower ICG doses preferentially kill neuroblastoma cells over fibroblast cells. [ABSTRACT FROM AUTHOR]

An unexpected high sensitivity to radiation has recently been discovered in a new generation of ultralow loss pure‐silica core optical fiber, the Corning Vascade EX1000. It is investigated how the injected power level of the probing signal at 1310 and 1550 nm affects the radiation‐induced attenuation (RIA) during and after steady‐state X‐ray exposure up to a total ionizing dose (TID) of 250 kGy (SiO2) at room temperature. The experiments confirm this fiber's high sensitivity to radiations, mainly due to the generation of metastable defects absorbing in the infrared domain. RIA kinetics are characterized by a non‐monotonic behavior, reaching a maximum of the radiation‐induced losses at 3.3 kGy (SiO2). Through successive radiation tests, the RIA dependence on the signal power level at the dose corresponding to the maximum of the RIA is studied, finding out that, for the two investigated wavelengths, the RIA follows a power law dependence in the 100 nW–1 mW injected power range, whose exponent seems independent on the dose rate (between 1 and 6 Gy s−1). The results highlight a clear photobleaching phenomenon, with the RIA associated to these metastable defects strongly reduced at higher injected powers of mW levels. [ABSTRACT FROM AUTHOR]

Photodynamic therapy (PDT) is an alternative treatment for the control of malignant diseases. It is based on the action of a photosensitizer (PS) molecule, which, upon being excited by light in a determined wavelength, reacts with oxygen and generates reactive oxygen species in target tissues, leading to cell death. We perform the non-covalent binding of PS’s with folic acid (FA) that have specific interactions with receptors, which are overexpressed on tumor cells to improve the targeted nature of PDT. The photobleaching of complexes and their components also was studied. The study showed that PS’s were actively bonded with FA non-covalently, with the molar ratio of components up to [FA]/ [PS] = 10.8, depending on the structure of the PS. As the duration of illumination increases, the photostability of the samples decreases. 20 % glycerin had a stabilizing effect on all samples. Therefore, the presence of glycerin in the solution is advisable to obtain stable non-covalent complexes. [ABSTRACT FROM AUTHOR]

Dinoflagellates in the family Symbiodiniaceae contain a number of species and play an important role in the establishment of coral reef ecosystems in oligotrophic marine waters. Effrenium voratum is likely an exclusively free-living and heterotrophic species of Symbiodiniaceae. How this species responds and acclimates to warming is largely unknown. The present study experimentally established the phenotypic landscapes related to the photobleaching and recovery processes of Effrenium voratum SCS01 following thermal stress. We found that thermal stress bleached the plastids of E. voratum SCS01 and caused the cells to become lighter in color. Thereafter, the bleached cells recovered rapidly when they returned to the optimal temperature. The dominant life form of E. voratum SCS01 shifted from mastigote cells to coccoid cells then returned to mastigote cells. Transcriptome analysis revealed that the photobleaching of E. voratum SCS01 was due to increased degradation and decreased biosynthesis of photosynthetic pigments. The thermally induced life form changes were related to the downregulation of genes for cell motility. Our results revealed the mechanism of photobleaching in E. voratum SCS01 and indicated life form transformation as a newly identified survival strategy of Effrenium voratum SCS01 under thermal stress.

Stable subphthalocyanine-type dyes with a high electron affinity attract much attention as potential substitutes for traditional fullerenes in molecular electronics devices. One possible way to enhance the acceptor properties of the subphthalocyanine core is by replacing the peripheral benzene fragments (C6H4) with 1,2,5-thiadiazole fragments (C2N2S1). However, the resistance of these materials to light or atmospheric effect remains an open question, which limits their further application in device manufacturing. In this work, we compare vacuum-deposited films of three derivatives, SubPzS3H0 (all peripheral fragments are 1,2,5-thiadiazoles), SubPzS2H4 (two fragments are 1,2,5-thiadiazoles and one fragment is benzene), and SubPcS0H12 (all benzenes, i.e., parent subphthalocyanine). Practically relevant substrates were used for deposition, namely, bare glass, glass/ITO or FTO, and PET/ITO. Photobleaching of films under continuous 1 sun illumination was studied in laboratory air, synthetic air, and ultrapure argon. It is shown that the exclusion of near-UV photons from the incident light spectrum, which corresponds to the absorption of subphthalocyanines in the Soret-band, strongly inhibits degradation. Absorption in the Q-band range initiates soft annealing rather than photobleaching of films. The stability of the films deposited on glass decreases as SubPzS3H0 > SubPzS2H4 > SubPcS0H12 in air, and vice versa in argon. The substrate adds more complexity to this picture. In argon, the ITO coating reduces degradation of all of the compounds equally, in contrast to the glass samples, while in air, the SubPzS3H0 films discolor completely. The latter reaction proceeds due to the indium-containing species migrating from the conductive coating.

European beech is one of the most important deciduous tree species in natural forest ecosystems in Central Europe. Its dominance is now being questioned by the emerging drought damages due to the increased incidence of severe summer droughts. In Switzerland, Fagus sylvatica have been observed in the Intercantonal Forest Observation Program since 1984. The dataset presented here includes 179176 annual observations of beech trees on 102 plots during 37 years. The plots cover gradients in drought, nitrogen deposition, ozone, age, altitude, and soil chemistry. In dry regions of Switzerland, the dry and hot summer of 2018 caused a serious branch dieback, increased mortality in Fagus sylvatica and increased yellowing of leaves. Beech trees recovered less after 2018 than after the dry summer 2003 which had been similar in drought intensity except that the drought in 2018 started earlier in spring. Our data analyses suggest the importance of drought in subsequent years for crown transparency and mortality in beech. The drought in 2018 followed previous dry years of 2015 and 2017 which pre-weakened the trees. Our long-term data indicate that the drought from up to three previous years were significant predictors for both tree mortality and for the proportion of trees with serious (>60%) crown transparency. The delay in mortality after the weakening event suggests also the importance of weakness parasites. The staining of active vessels with safranine revealed that the cavitation caused by the low tree water potentials in 2018 persisted at least partially in 2019. Thus, the ability of the branches to conduct water was reduced and the branches dried out. Furthermore, photooxidation in light-exposed leaves has increased strongly since 2011. This phenomenon was related to low concentrations of foliar phosphorus (P) and hot temperatures before leaf harvest. The observed drought effects can be categorized as (i) hydraulic failure (branch dieback), (ii) energy starvation as a consequence of closed stomata and P deficiency (photooxidation) and (iii) infestation with weakness parasites (beech bark disease and root rots).

Due to their large contact and loading surfaces as well as high sensitivities to chemical changes, graphene‐based materials (GBMs) are increasingly being employed into novel nanomedicine technologies. Here biomolecule—monolayer graphene—kidney tissue hybrid structures are studied using mapping micro‐Raman and fluorescence spectroscopies. Because in this configuration graphene interacts with molecules on both sides, a double‐sided graphene‐enhanced Raman scattering (GERS) effect up to ≈10.1 is found for biomolecules adsorbed on graphene and amino acids in the kidney tissue below graphene. Moreover, graphene causes an efficient autofluorescence quenching (FLQ) up to ≈20% emitted by the kidney tissue. Despite the complexity of such layered materials, the intriguing simultaneous occurrence of double‐sided GERS (a new development of GERS) and FLQ phenomena can be well explained by suitable molecular structure and energy level alignment between molecules and graphene. These result in effective charge transfer mediated by non‐covalent interactions as indicated by correlative strain, doping, and defect analyses in graphene based on the Raman data and energy level calculations. Last, the advantages of using graphene over standard photobleaching are demonstrated. This work can be extended to other macromolecular entities toward integrating GBMs in versatile drug delivery, imaging, and sensing devices. [ABSTRACT FROM AUTHOR]