Search

The rapid photobleaching of near-infrared (NIR) dye-sensitized upconversion nanosystems is one of the crucial problems that has blocked their technological applications. Uncovering the photophysical and photochemical pathways of NIR dyes would help to elucidate the photobleaching mechanism and thereby improve the photostability of the system. Here we investigate the triplet dynamics of NIR dyes and their interaction with triplet oxygen in the typically investigated IR806-sensitized upconversion nanoparticle (UCNP) nanosystem. Low-temperature fluorescence at 77 K provides direct proof of the generation of singlet oxygen (1O2) under 808 nm laser irradiation. Mass spectrometry indicates that all three double bonds in the structure of IR806 can be broken in the photochemical process. Coupling IR806 to the surface of UCNPs can accelerate its triplet dynamics, thus producing more 1O2 to photocleave IR806. Importantly, we find that the addition of β-carotene can scavenge the generated 1O2, thereby providing a simple method to effectively inhibit photobleaching.

Macrophage polarization in neurotoxic (M1) or neuroprotective (M2) phenotypes is known to play a significant role in neuropathic pain, but its behavioral dynamics and underlying mechanism remain largely unknown. Two‐photon excitation microscopy (2PEM) is a promising functional imaging tool for investigating the mechanism of cellular behavior, as using near‐infrared excitation wavelengths is less subjected to light scattering. However, the higher‐order photobleaching effect in 2PEM can seriously hamper its applications to long‐term live‐cell studies. Here, we demonstrate a GHz femtosecond (fs) 2PEM that enables hours‐long live‐cell imaging of macrophage behavior with reduced higher‐order photobleaching effect—by leveraging the repetition rate of fs pulses according to the fluorescence lifetime of fluorophores. Using this new functional 2PEM platform, we measure the polarization characteristics of macrophages, especially the long‐term cellular behavior in efferocytosis, unveiling the dynamic mechanism of neuroprotective macrophage polarization in neuropathic pain. These efforts can create new opportunities for understanding long‐term cellular dynamic behavior in neuropathic pain, as well as other neurobiological problems, and thus dissecting the underlying complex pathogenesis. [ABSTRACT FROM AUTHOR]

The epipelagic macroalgae of Ulva prolifera and Sargassum are the primary contributors to widespread seaweed tides globally. Both ocean plants release large amounts of chromophoric dissolved organic matter (CDOM) into the surrounding seawater. The photochemical reactivity of this CDOM, however, has not been adequately addressed. In this study, we extracted CDOM from Ulva prolifera and Sargassum, examined their ultraviolet (UV)-visible absorption characteristics, and quantified their broadband apparent quantum yields (AQY) of absorbance photobleaching and photomineralization (in terms of CO2, CO, and CH4 photoproduction). On a per-unit-weight basis, Sargassum leached 3.5 times more CDOM than did Ulva prolifera in terms of the absorption coefficient averaged over 254-500 nm. Both Ulva prolifera and Sargassum CDOM were characterized by quasi-exponential decay absorption spectra, with Sargassum CDOM exhibiting a distinct shoulder over 310-350 nm suggestive of mycosporine amino acids. The Sargassum CDOM had a higher photobleaching AQY but lower photomineralization AQYs compared to Ulva prolifera CDOM. The photobleaching and photomineralization AQYs of both macroalgal CDOM are, however, orders of magnitude higher than those of CDOM in various natural waters. Potential photoproduction rates of CO2 and CO from the Ulva prolifera CDOM and Sargassum CDOM during the bloom periods are several times to orders of magnitude higher than the air-sea fluxes of these gases in the absence of the macroalgae. This study demonstrates that CDOM released by Ulva prolifera and Sargassum is extremely prone to photobleaching and photomineralization, rendering floating mats of these plants in oceans as potential "hotspots" of greenhouse gas emissions to the atmosphere. This photochemical feedback should be considered when assessing ocean afforestation as a CO2 removal approach to mitigate climate warming. [ABSTRACT FROM AUTHOR]

Imaging artifacts often hinder the accuracy of microscopy images, affecting sample analyses and their interpretations. In this article, we explore five prominent imaging artifacts that commonly plague microscopy: blooming/saturation, pixelation, temporal/time varying noise, phototoxicity/photobleaching, and compression. Effective mitigation of these imaging artifacts hinges on careful consideration of key camera parameters, including quantum efficiency, bit-depth, resolution, and dynamic range, each crucial for optimizing image quality and fidelity. A wide range of camera solutions offered by e-con Systems provides consistent support to the various types of microscopy techniques from fluorescence microscopy to live cell imaging. [ABSTRACT FROM AUTHOR]

Herein, we report on the antimicrobial photodynamic effect of polymeric nanoparticles containing the endogenous photosensitizer protoporphyrin IX. Compared to equivalent doses of the free photosensitizer, we demonstrated that the photodynamic antimicrobial efficacy of PLGA (polylactic-co-glycolic acid) nanoparticles containing protoporphyrin IX (PpIX) against pathogenic Staphylococcus aureus (S. aureus) is preserved after encapsulation, while photobleaching is reduced. In addition, compared to equivalent doses of the free porphyrin, we show that a reduction in the cytotoxicity in mammalian cell cultures is observed when encapsulated. Therefore, the encapsulation of protoporphyrin IX reduces its photodegradation, while the released photosensitizer maintains its ability to generate reactive oxygen species upon light irradiation. The polymeric nanoencapsulation promotes aqueous solubility for the hydrophobic PpIX, improves its photostability and reduces the cytotoxicity, while providing an extended release of this endogenous photosensitizer. [ABSTRACT FROM AUTHOR]

Photodynamic therapy (PDT) is already used to treat many cancers, including breast cancer, the most common cancer in women worldwide. The destruction basis of this method is on produced singlet oxygen which is extremely reactive and is a major agent of tumor cell killing. The measurement of singlet oxygen produced within PDT is essential in predicting treatment outcomes and their optimization. This study aims to determine the optimal total light dose administered during PDT by calculating the singlet oxygen to facilitate the prediction of the treatment outcome in mice bearing 4T1 cell breast cancer. Monitoring the changes in photosensitizer fluorescence signals during PDT due to photobleaching can be one of the methods of determination of singlet oxygen generation in the PDT process. This study determined the oxygen singlet as a photodynamic dose from the three‐dimensional Monte Carlo method and the photobleaching empirical dose constant. The photobleaching dose constant was established non‐invasively by monitoring the in vivo protoporphyrin IX (PpIX) fluorescence and photobleaching during PDT. The photobleaching dose constant (β) in J/cm2 was calculated using empirical fluorescence data. The in vivo photobleaching dose constant of aminolevulinic acid was found to be 11.6 J/cm2 and based on this value, the optimal treatment light dose was estimated at 120 J/cm2 in mice bearing 4T1 breast cancer. It is concluded that information can be obtained regarding optimal treatment parameters by monitoring the in vivo PpIX fluorescence and photobleaching during PDT. [ABSTRACT FROM AUTHOR]

While organic photocatalysts provide increasingly versatile chemical pathways under mild conditions, their long‐term stability remains understudied. Here, the photobleaching behavior of xanthene dye photocatalysts is investigated. Rose Bengal, Eosin Y, and fluorescein are studied when in solution, when grafted to glass beads, and when incorporated into polymer brushes that are tethered to glass beads. This provides a comparison between xanthene's stability as a homogeneous and as a heterogeneous photocatalyst. Photobleaching is investigated using UV–vis, diffuse reflectance UV–vis (DR UV–vis), and fluorescence microscopy. Xanthene dyes as homogeneous photocatalysts exhibit the highest photostability, while the grafted systems appeared to fade more rapidly. Notably, heterogenization appears to have different effects based on the photocatalyst system, and further altering the photocatalyst environment with reagents may improve stability. [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. [ABSTRACT FROM AUTHOR]

Herein, we report on the antimicrobial photodynamic effect of polymeric nanoparticles containing the endogenous photosensitizer protoporphyrin IX. Compared to equivalent doses of the free photosensitizer, we demonstrated that the photodynamic antimicrobial efficacy of PLGA (polylactic-co-glycolic acid) nanoparticles containing protoporphyrin IX (PpIX) against pathogenic Staphylococcus aureus (S. aureus) is preserved after encapsulation, while photobleaching is reduced. In addition, compared to equivalent doses of the free porphyrin, we show that a reduction in the cytotoxicity in mammalian cell cultures is observed when encapsulated. Therefore, the encapsulation of protoporphyrin IX reduces its photodegradation, while the released photosensitizer maintains its ability to generate reactive oxygen species upon light irradiation. The polymeric nanoencapsulation promotes aqueous solubility for the hydrophobic PpIX, improves its photostability and reduces the cytotoxicity, while providing an extended release of this endogenous photosensitizer.

[Figure: see text].

Quenching singlet oxygen is the key to improve photostability of fluorescent dyes. We have systematically studied the capability of a new singlet oxygen quencher, DABCOnium (an alkylated derivative of 1,4‐diazabicyclo[2.2.2] octane) on removing singlet oxygen. Results showed that DABCOnium can significantly enhancing photostability of fluorescent thin films for more than 4.3 times compared with that without DABCOnium. After 2 hours of continuous irradiation, fluorescent film doped with DABCOnium retained 90 % of its original fluorescence intensity, which paves a new way in designing high performance singlet oxygen quenchers. [ABSTRACT FROM AUTHOR]

Inorganic glasses doped with luminescent silver clusters are promising materials for photonic applications as white light generation, optical data storage, and spectral conversion. This work reports the photostability study of luminescent silver clusters dispersed in silica‐based glass under continuous ultraviolet irradiation. The photobleaching process model is proposed and the quantum yield of photobleaching is derived from the experimental data. The proposed mechanism of photobleaching is photoionization of silver clusters. Degradation of cluster luminescence is reversible and restores after the heat treatment, indicating the possibility to release trapped electrons and return the initial charge state of clusters. The effect of heat treatment temperature on the luminescence restoration is studied, the amount of restored luminescent clusters depends linearly on the heat treatment temperature. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Recent results obtained in our experimental investigation on the influence of the illumination with continuous wave (c.w.) laser light at 532 nm on the transmittance at this wavelength and on the photoluminescence of the DNA-CTMA-RhB in butanol compound are reported. The temporal dependence of absorption changes induced by c. w. light at 532 nm in DNA-CTMA-RhB in butanol solution, and, for comparison, in RhB in butanol solution, is investigated experimentally and analyzed in order to assess the effect of DNA-CTMA on this light-induced process. The evolution in time of the peak's amplitude and wavelength of the photoluminescence spectra in solutions of DNA-CTMA-RhB in butanol, during their excitation with laser light at 532 nm wavelength has been also investigated and discussed.

Due to the increased application of lasers in different fields (industry, medicine, etc.), there is a growing need for new laser sources with good beam quality and variable emission wavelength. At the same time, for environmental reasons, the obtaining of novel eco-friendly active optical materials, such as those based on the deoxyribonucleic acid (DNA) biopolymer, with optimal light emission properties, is of high interest. The results obtained in this study of the temporal dependence of the transmittance and of the light emission in thin films of DNA–CTMA–Rhodamine 610 (at different Rhodamine concentrations) (DNA–CTMA–Rh610), when they are illuminated with continuous wave laser light at 532 nm (frequently used in the optical pumping of dye lasers), are presented and discussed. The transmittance results obtained for thin film samples are compared to those obtained for the DNA–CTMA–Rh610 solutions in butanol, from which the films have been made, and also with those obtained for Rh610 solutions in butanol with the same concentrations. The investigation was performed in order to assess the influence of the DNA-CTMA and of the green laser light at 532 nm wavelength on relevant chromophore properties such as light transmission and fluorescence emission. The results obtained revealed that the DNA–CTMA matrix has an active influence on the Rhodamine 610 emission, in the whole range of concentrations of the investigated samples. [ABSTRACT FROM AUTHOR]

The genetic alterations contributing to migration proficiency, a phenotypic hallmark of metastatic cells required for colonizing distant organs, remain poorly defined. Here, we used single-cell magneto-optical capture (scMOCa) to isolate fast cells from heterogeneous human breast cancer cell populations, based on their migratory ability alone. We show that captured fast cell subpopulations retain higher migration speed and focal adhesion dynamics over many generations as a result of a motility-related transcriptomic profile. Upregulated genes in isolated fast cells encoded integrin subunits, proto-cadherins and numerous other genes associated with cell migration. Dysregulation of several of these genes correlates with poor survival outcomes in people with breast cancer, and primary tumors established from fast cells generated a higher number of circulating tumor cells and soft tissue metastases in pre-clinical mouse models. Subpopulations of cells selected for a highly migratory phenotype demonstrated an increased fitness for metastasis. [ABSTRACT FROM AUTHOR]

Trace metals are essential elements that play key roles in a number of biochemical processes governing human visual physiology in health and disease. Several trace metals, such as zinc, have been shown to play important roles in the visual phototransduction process. In spite of this, there has been little research conducted on the direct effect of trace metal elements on the visual photoreceptor rhodopsin. In the current study, we have determined the effect of several metal ions, such as iron, copper, chromium, manganese, and nickel, on the conformational stability of rhodopsin. To this aim, we analyzed, by means of UV-visible and fluorescence spectroscopic methods, the effects of these trace elements on the thermal stability of dark rhodopsin, the stability of its active Metarhodopsin II conformation, and its chromophore regeneration. Our results show that copper prevented rhodopsin regeneration and slowed down the retinal release process after illumination. In turn, Fe3+, but not Fe2+, increased the thermal stability of the dark inactive conformation of rhodopsin, whereas copper ions markedly decreased it. These findings stress the important role of trace metals in retinal physiology at the photoreceptor level and may be useful for the development of novel therapeutic strategies to treat retinal disease. [ABSTRACT FROM AUTHOR]

We investigated photo-induced effects in Ge-Ga-Se films. It was found that, thermal annealing causes the increase of the bandgap in the as-prepared films, and all three annealed films show photodarkening (PD) behavior with light illumination but the change of optical bandgap is minimum in Ge25 film. Such PD behavior can be recovered by reannealing of the film. The kinetic process of Tf/Ti exhibits part recovering of PD during the cycle of laser on and off. Moreover, no obvious structural change can be observed in the Raman spectra of the films at different states, especially in that of Ge25 film. [ABSTRACT FROM AUTHOR]

Determining the axial position of an emitter with nanoscale precision is critical to a fundamental imaging methodology. While there are many advanced optical techniques being applied to high-resolution imaging, high-axial-resolution topography imaging of living cells is particularly challenging. Here, we present an application of metamaterial-assisted photobleaching microscopy (MAPM) with high-axial resolution to characterize morphological properties of living cells. Quantitative imaging of changes in the morphology of live cells is obtained by topographic and statistical analysis. The time-lapse topography image using the metamaterial-induced photostability provides information about growth factor induced changes in the cell morphology with high-axial resolution.

Photodynamic therapy (PDT) is recognized as a powerful method to inactivate cells. However, the photosensitizer (PS), a key component of PDT, has suffered from undesired photobleaching. Photobleaching reduces reactive oxygen species (ROS) yields, leading to the compromise of and even the loss of the photodynamic effect of the PS. Therefore, much effort has been devoted to minimizing photobleaching in order to ensure that there is no loss of photodynamic efficacy. Here, we report that a type of PS aggregate showed neither photobleaching nor photodynamic action. Upon direct contact with bacteria, the PS aggregate was found to fall apart into PS monomers and thus possessed photodynamic inactivation against bacteria. Interestingly, the disassembly of the bound PS aggregate in the presence of bacteria was intensified by illumination, generating more PS monomers and leading to an enhanced antibacterial photodynamic effect. This demonstrated that on a bacterial surface, the PS aggregate photo-inactivated bacteria via PS monomer during irradiation, where the photodynamic efficiency was retained without photobleaching. Further mechanistic studies showed that PS monomers disrupted bacterial membranes and affected the expression of genes related to cell wall synthesis, bacterial membrane integrity, and oxidative stress. The results obtained here are applicable to other types of PSs in PDT. [ABSTRACT FROM AUTHOR]

Performing multi-color nanoscopy for extended times is challenging due to the rapid photobleaching rate of most fluorophores. Here we describe a new fluorophore (Yale-595) and a bio-orthogonal labeling strategy that enables two-color super-resolution (STED) and 3D confocal imaging of two organelles simultaneously for extended times using high-density environmentally sensitive (HIDE) probes. Because HIDE probes are small, cell-permeant molecules, they can visualize dual organelle dynamics in hard-to-transfect cell lines by super-resolution for over an order of magnitude longer than with tagged proteins. The extended time domain possible using these tools reveals dynamic nanoscale targeting between different organelles.

The association of plasma membrane (PM)-localized voltage-gated potassium (Kv2) channels with endoplasmic reticulum (ER)-localized vesicle-associated membrane protein-associated proteins VAPA and VAPB defines ER-PM junctions in mammalian brain neurons. Here, we used proteomics to identify proteins associated with Kv2/VAP-containing ER-PM junctions. We found that the VAP-interacting membrane-associated phosphatidylinositol (PtdIns) transfer proteins PYK2 N-terminal domain-interacting receptor 2 (Nir2) and Nir3 specifically associate with Kv2.1 complexes. When coexpressed with Kv2.1 and VAPA in HEK293T cells, Nir2 colocalized with cell-surface-conducting and -nonconducting Kv2.1 isoforms. This was enhanced by muscarinic-mediated PtdIns(4,5)P2 hydrolysis, leading to dynamic recruitment of Nir2 to Kv2.1 clusters. In cultured rat hippocampal neurons, exogenously expressed Nir2 did not strongly colocalize with Kv2.1, unless exogenous VAPA was also expressed, supporting the notion that VAPA mediates the spatial association of Kv2.1 and Nir2. Immunolabeling signals of endogenous Kv2.1, Nir2, and VAP puncta were spatially correlated in cultured neurons. Fluorescence-recovery-after-photobleaching experiments revealed that Kv2.1, VAPA, and Nir2 have comparable turnover rates at ER-PM junctions, suggesting that they form complexes at these sites. Exogenous Kv2.1 expression in HEK293T cells resulted in significant differences in the kinetics of PtdIns(4,5)P2 recovery following repetitive muscarinic stimulation, with no apparent impact on resting PtdIns(4,5)P2 or PtdIns(4)P levels. Finally, the brains of Kv2.1-knockout mice had altered composition of PtdIns lipids, suggesting a crucial role for native Kv2.1-containing ER-PM junctions in regulating PtdIns lipid metabolism in brain neurons. These results suggest that ER-PM junctions formed by Kv2 channel-VAP pairing regulate PtdIns lipid homeostasis via VAP-associated PtdIns transfer proteins.

Abstract 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.

Viscoelastic properties are one of the most fundamental properties of chiral liquid crystals. In general, their determination is not a straightforward task. The main problem is the multitude of physical parameters needed to determine the value of the elasticity and viscosity constants. It is also necessary to consider the character of a respective phase. This problem is particularly important in the case of chiral phases such as ferroelectric and antiferroelectric phases or in the blue phases. There are several experimental methods to measure viscosity and elasticity constants in chiral phases. These methods use various phenomena to detect deformation, e.g., light transmission, polarization current, light modulation, dielectric constant and helix deformation or helix unwinding. Commonly, an external electric field is used to induce deformation, the homogeneity of which inside the cell is essential. This study is focused on the analysis of the effect of laser photobleaching on the electro-optic properties of the antiferroelectric liquid crystal and on the homogeneity of the electric field. The results obtained by confocal microscopy as a function of the cell depth are presented. The influence of the stabilization procedure of the isolated region performed by controlled laser photobleaching on the electro-optic properties has been studied. The observation was conducted using a polarizing microscope, and numerical analysis of two-dimensional colored textures was performed. The obtained results suggest that laser photobleaching can produce an anchoring effect, which has a positive effect on the electro-optic properties of antiferroelectric liquid crystal. [ABSTRACT FROM AUTHOR]

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]

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).

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.

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.

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]

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]

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.

Due to the increased application of lasers in different fields (industry, medicine, etc.), there is a growing need for new laser sources with good beam quality and variable emission wavelength. At the same time, for environmental reasons, the obtaining of novel eco-friendly active optical materials, such as those based on the deoxyribonucleic acid (DNA) biopolymer, with optimal light emission properties, is of high interest. The results obtained in this study of the temporal dependence of the transmittance and of the light emission in thin films of DNA–CTMA–Rhodamine 610 (at different Rhodamine concentrations) (DNA–CTMA–Rh610), when they are illuminated with continuous wave laser light at 532 nm (frequently used in the optical pumping of dye lasers), are presented and discussed. The transmittance results obtained for thin film samples are compared to those obtained for the DNA–CTMA–Rh610 solutions in butanol, from which the films have been made, and also with those obtained for Rh610 solutions in butanol with the same concentrations. The investigation was performed in order to assess the influence of the DNA-CTMA and of the green laser light at 532 nm wavelength on relevant chromophore properties such as light transmission and fluorescence emission. The results obtained revealed that the DNA–CTMA matrix has an active influence on the Rhodamine 610 emission, in the whole range of concentrations of the investigated samples.

Trace metals are essential elements that play key roles in a number of biochemical processes governing human visual physiology in health and disease. Several trace metals, such as zinc, have been shown to play important roles in the visual phototransduction process. In spite of this, there has been little research conducted on the direct effect of trace metal elements on the visual photoreceptor rhodopsin. In the current study, we have determined the effect of several metal ions, such as iron, copper, chromium, manganese, and nickel, on the conformational stability of rhodopsin. To this aim, we analyzed, by means of UV-visible and fluorescence spectroscopic methods, the effects of these trace elements on the thermal stability of dark rhodopsin, the stability of its active Metarhodopsin II conformation, and its chromophore regeneration. Our results show that copper prevented rhodopsin regeneration and slowed down the retinal release process after illumination. In turn, Fe3+, but not Fe2+, increased the thermal stability of the dark inactive conformation of rhodopsin, whereas copper ions markedly decreased it. These findings stress the important role of trace metals in retinal physiology at the photoreceptor level and may be useful for the development of novel therapeutic strategies to treat retinal disease.

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.

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 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]

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]

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]