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Whole-mount in situ hybridization (WISH) is a widely used method that supports the concept of "seeing is believing" by enabling the visualization of gene expression patterns in whole-mount multicellular samples or sections. This technique is essential in the study of epimorphic regeneration in cold-blooded vertebrates, where complex three-dimensional organs such as tails, limbs, and eyes are completely restored after loss. The tadpoles of the frog X. laevis serve as a convenient model for studying regeneration, as they can regenerate their tails within a week after amputation. Modern high-throughput sequencing methods have identified various cell populations involved in the regeneration process and determined the repertoire of genes activated during this time. Specifically, a population of reparative myeloid cells expressing mmp9 as a marker gene has been shown to be crucial for the initial stages of tail regeneration in X. laevis tadpoles. The validation of these data and further examination using WISH offers the advantage of providing detailed information on the spatial and temporal dynamics of target gene expression levels. However, detecting mRNA by WISH can be challenging when mRNA levels are very low, transcripts are localized in hard-to-access areas, or tissue samples are prone to background staining, as is the case with X. laevis regenerating tail samples. Here, we describe additional treatments for regenerating tail samples that minimize background staining and enhance the visualization of cells containing target RNA through in situ hybridization. Using an optimized WISH protocol on X. laevis tadpole tail regenerates, we obtained novel data on the mmp9 expression pattern during the first day post-amputation at the regeneration-competent stage 40 and the regeneration-incompetent stage 47 (refractory period). The significant differences in the expression patterns indicate that mmp9 activity is positively correlated with regeneration competence. [ABSTRACT FROM AUTHOR]

Advancements in cellular imaging have significantly enhanced our understanding of membrane potential and Ca2⁺ dynamics, which are crucial for various cellular processes. Voltage-sensitive dyes (VSDs) are pivotal in this field, enabling non-invasive, high-resolution visualization of electrical activity in cells. This review discusses the various types of VSDs, including electrochromic, Förster Resonance Energy Transfer (FRET)-based, and Photoinduced Electron Transfer (PeT)-based dyes. VSDs are essential tools for studying mitochondrial activity and neuronal function and are frequently used in conjunction with Ca2⁺ indicators to elucidate the complex relationship between membrane potential and Ca2⁺ fluxes. The development of novel dyes with improved photostability and reduced toxicity continues to expand the potential of VSDs in biomedical research. This review underscores the importance of VSDs in advancing our understanding of cellular bioenergetics, signaling, and disease mechanisms. [ABSTRACT FROM AUTHOR]

Fluorofluorophores are a unique class of fluorophores that can be solubilized in perfluorocarbons (PFCs) and used to study biological systems. However, because of the low dielectric constant and high oxygen solubility in the fluorous phase, the brightness and photostability of the fluorofluorophores are significantly diminished. Here, we leveraged the tight ion pairing in the fluorous phase to improve the photophysical properties of a fluorous soluble pentamethine dye (FCy5) via counterion exchange. We found that larger, softer, fluorinated, aryl borate counterions promote the ideal polymethine state where charge delocalization across the polymethine chain increases the brightness (6‐fold) and photostability (55‐fold) of FCy5. [ABSTRACT FROM AUTHOR]

This study details best practices for high-content screening (HCS) using the cell painting (CP) technique to enhance drug discovery and biomedical research. HCS, an automated fluorescence microscopy method, offers a detailed analysis of cellular responses by capturing multidimensional data on cellular morphology, protein expression, localization, and function. The CP technique involves staining cells with specific fluorescent dyes and analyzing them with high-resolution fluorescence microscopy. It is particularly effective in generating drug-specific phenotypic fingerprints or "phenoprints," providing deeper insights into drug mechanisms. Strategies to enhance CP performance include using water immersion and high NA (numerical aperture) objective lenses, large sCMOS detectors, and confocal imaging with maximum intensity projection (MIP) imaging. These strategies are pivotal for producing high-quality images for accurate morphological analysis. This study addresses some of the challenges in standardizing image and data acquisition in CP and identifies optimal acquisition parameters using the Yokogawa CellVoyager CV8000. The results emphasize the critical role of optimizing the signal-to-background ratio in fluorescence imaging by adjusting laser power and camera exposure time. This optimization is essential for enhancing signal detection, reducing background noise and photobleaching, and improving image analysis and reproducibility from experiment to experiment. By refining data acquisition practices and utilizing sophisticated imaging technologies, one can achieve superior cellular feature data, leading to more precise and reliable analyses in drug discovery and disease modeling. [ABSTRACT FROM AUTHOR]

Understanding the spatial distribution of key cell populations is critical in advancing biomedical research and the development of novel therapeutics. Highly multiplexed biomarker analysis is achieved with single-cell spatial context on the CellScape, a microscopy platform that enables quantitative spatial phenotyping of entire tissue sections mounted on standard histology slides. Leveraging high-resolution, high dynamic range imaging, and automated reagent delivery, the CellScape represents an attractive platform for spatial biology researchers from discovery to translation. [ABSTRACT FROM AUTHOR]

Unimolecular Type II radical photoinitiators (PIs) have received significant attention in photocuring owing to the fact that they improve the sustainability of the overall process compared with traditional Type II radical photoinitiators. However, the photopolymerization efficiency of unimolecular Type II radical photoinitiators is hindered by their short excitation wavelengths, poor photon capture abilities, and inefficient photobleaching performance. Herein, we report a coumarin-based self-sufficient initiator (C-NA), which is designed by integrating "hydrogen donor" and "hydrogen acceptor" into the coumarin framework and used for single-component visible light curing. C-NA exhibits a visible light absorbance and high molar extinction coefficient and is completely photobleached under the irradiation of 405 nm light-emitting diodes (LEDs). The formation of free radicals arises from the transfer of hydrogen from the diethylamino group to the coumarin framework, together with a highly efficient photodegradation process of C-NA. Finally, C-NA was successfully applied to prepare a transparent film material. Therefore, C-NA offers new insights into the design of promising unimolecular Type II radical photoinitiators for photocuring. [ABSTRACT FROM AUTHOR]

Advancements in cellular imaging have significantly enhanced our understanding of membrane potential and Ca2⁺ dynamics, which are crucial for various cellular processes. Voltage-sensitive dyes (VSDs) are pivotal in this field, enabling non-invasive, high-resolution visualization of electrical activity in cells. This review discusses the various types of VSDs, including electrochromic, Förster Resonance Energy Transfer (FRET)-based, and Photoinduced Electron Transfer (PeT)-based dyes. VSDs are essential tools for studying mitochondrial activity and neuronal function and are frequently used in conjunction with Ca2⁺ indicators to elucidate the complex relationship between membrane potential and Ca2⁺ fluxes. The development of novel dyes with improved photostability and reduced toxicity continues to expand the potential of VSDs in biomedical research. This review underscores the importance of VSDs in advancing our understanding of cellular bioenergetics, signaling, and disease mechanisms.

Strong light–matter interactions have received a lot of attention, for example in the pursuit of plasmonic-excitonic structures as coherent light sources with low-power threshold. In this study, we investigate the influence of room temperature strong coupling between surface plasmon polaritons (SPP) and excitons on fluorescence lifetimes and photobleaching effects. Our plasmonic-photonic structure, comprising of thin silver (Ag) and gold (Au) layers with a Rhodamine 6G (R6G) dye layer, shows a clear shift in the plasmon resonance and R6G absorption lines with varying incident angles, indicative of strong coupling, with a measured Rabi splitting of approximately 90 meV. Fluorescence lifetime imaging microscopy (FLIM) was then employed to assess photobleaching, revealing a significant reduction in photobleaching effect for in strongly coupled plasmonic-excitonic structures compared to single Rhodamine R6G layers. Our findings indicate the pivotal role of strong light–matter interactions in reducing photobleaching effects and stabilizing fluorescence intensities, offering promising avenues for developing quantum multiparticle nanophotonic devices with enhanced stability and performance.

Physalis grisea has significant medicinal properties and its edible fruits have substantial economic value. Virus-induced gene silencing (VIGS) is a highly effective and rapid method for assessing the function of plant genes. While various VIGS have been successfully utilized in plants, there are no reports of VIGS systems targeting P. grisea so far. In this study, the establishment of an efficient and stable Tobacco Rattle Virus (TRV)-mediated VIGS system was investigated in P. grisea. Factors influencing VIGS activity were compared, including carrier length, acetosyringone (AS) concentration, optical density, the inoculation method of Agrobacterium tumefaciens, and processing temperature. As a result, two TRV-based VIGS systems were successfully developed. The first system employs a leaf infiltration method with an acetosyringone concentration of 0.2 µmol/L and an optical density (OD₆₀₀) of 1.0, utilizing TRV2-PDS436 at 20℃. The second system involves a stem infiltration while maintaining the same temperature, vector insert length, AS concentration, and OD₆₀₀. The silencing efficiencies were recorded at 83.34% and 56.40%, respectively, demonstrating the potential of these systems for research in P. grisea. This method provides a rapid and effective method to analyze the function of inverted genes, helps to elucidate unknown gene functions in P. grisea, and provides new avenues for genetic and functional research on this species.Key message: The PgPDS gene serves as a marker in this study, which employed various carrier lengths, different concentrations of infectious liquids, diverse penetration methods, and a range of processing temperatures to conduct high-efficiency VIGS research on P. grisea. [ABSTRACT FROM AUTHOR]

Protection against the negative effects of solar radiation involves using cosmetics with a UV filter, but visible radiation can also have negative effects. We use dietary supplements and take medications; unfortunately, many of them contain substances that degrade under the influence of visible light, which transform into chemical compounds harmful to health. Manufacturers often include information on the prohibition of exposure to sunlight on the packaging, but consumers often do not read the product leaflet. The solution to this problem may be the addition of silver particles to preparations. In the presented article, we proposed the use of silver nanoparticles to reduce the photobleaching and photoreaction of fluorophore, while increasing the fluorescence intensity. For our research, we used a compound that is particularly sensitive to radiation: hypericin. [ABSTRACT FROM AUTHOR]

We investigated the influence of the photobleaching (PB) effect on the dosimetry performances of a phosphosilicate single-mode optical fiber (core diameter of 6.6 µm) operated at 638 nm, within the framework of the LUMINA project. Different irradiation tests were performed under ~40 keV mean energy fluence X-rays at a 530 µ Gy(SiO2)/s dose rate to measure in situ the radiation-induced attenuation (RIA) growth and decay kinetics while injecting a 638 nm laser diode source with powers varying from 500 nW to 1 mW. For injected continuous power values under 1 µW, we did not measure any relevant influence of the photobleaching effect on the fiber radiation sensitivity coefficient of ~140 dB km−1 Gy−1 up to ~30 Gy. Above 1 µW, the fiber radiation sensitivity is significantly reduced due to the PB associated with the signal and can decrease to ~80 dB km−1 Gy−1 at 1 mW, strongly affecting the capability of this fiber to serve as a dosimeter-sensitive element. Higher power values up to 50 µW can still be used by properly choosing a pulsed regime with periodic injection cycles to reduce the PB efficiency and maintain the dosimetry properties. Basing on the acquired data, a simple model of the photobleaching effect on a coil of the investigated fiber is proposed in order to estimate its sensitivity coefficient evolution as a function of the cumulated dose and its fiber length when injecting a certain laser power. Additional studies need to investigate the influence of the temperature and the dose rate on the PB effects since these parameters were fixed during all the reported acquisitions. [ABSTRACT FROM AUTHOR]

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.

Whole-mount in situ hybridization (WISH) is a widely used method that supports the concept of “seeing is believing” by enabling the visualization of gene expression patterns in whole-mount multicellular samples or sections. This technique is essential in the study of epimorphic regeneration in cold-blooded vertebrates, where complex three-dimensional organs such as tails, limbs, and eyes are completely restored after loss. The tadpoles of the frog X. laevis serve as a convenient model for studying regeneration, as they can regenerate their tails within a week after amputation. Modern high-throughput sequencing methods have identified various cell populations involved in the regeneration process and determined the repertoire of genes activated during this time. Specifically, a population of reparative myeloid cells expressing mmp9 as a marker gene has been shown to be crucial for the initial stages of tail regeneration in X. laevis tadpoles. The validation of these data and further examination using WISH offers the advantage of providing detailed information on the spatial and temporal dynamics of target gene expression levels. However, detecting mRNA by WISH can be challenging when mRNA levels are very low, transcripts are localized in hard-to-access areas, or tissue samples are prone to background staining, as is the case with X. laevis regenerating tail samples. Here, we describe additional treatments for regenerating tail samples that minimize background staining and enhance the visualization of cells containing target RNA through in situ hybridization. Using an optimized WISH protocol on X. laevis tadpole tail regenerates, we obtained novel data on the mmp9 expression pattern during the first day post-amputation at the regeneration-competent stage 40 and the regeneration-incompetent stage 47 (refractory period). The significant differences in the expression patterns indicate that mmp9 activity is positively correlated with regeneration competence.

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]

We investigated the influence of the photobleaching (PB) effect on the dosimetry performances of a phosphosilicate single-mode optical fiber (core diameter of 6.6 µm) operated at 638 nm, within the framework of the LUMINA project. Different irradiation tests were performed under ~40 keV mean energy fluence X-rays at a 530 µ Gy(SiO2)/s dose rate to measure in situ the radiation-induced attenuation (RIA) growth and decay kinetics while injecting a 638 nm laser diode source with powers varying from 500 nW to 1 mW. For injected continuous power values under 1 µW, we did not measure any relevant influence of the photobleaching effect on the fiber radiation sensitivity coefficient of ~140 dB km−1 Gy−1 up to ~30 Gy. Above 1 µW, the fiber radiation sensitivity is significantly reduced due to the PB associated with the signal and can decrease to ~80 dB km−1 Gy−1 at 1 mW, strongly affecting the capability of this fiber to serve as a dosimeter-sensitive element. Higher power values up to 50 µW can still be used by properly choosing a pulsed regime with periodic injection cycles to reduce the PB efficiency and maintain the dosimetry properties. Basing on the acquired data, a simple model of the photobleaching effect on a coil of the investigated fiber is proposed in order to estimate its sensitivity coefficient evolution as a function of the cumulated dose and its fiber length when injecting a certain laser power. Additional studies need to investigate the influence of the temperature and the dose rate on the PB effects since these parameters were fixed during all the reported acquisitions.

Background: The study is aimed at developing a method for monitoring photodynamic therapy (PDT) of a tumor using chlorin-type photosensitizers (PSs). Lack of monitoring of chlorin e6 (Cе6) photobleaching, hemoglobin oxygenation and blood flow during light exposure can limit the PDT effectiveness. Materials and methods: Phototheranostics includes spectral-fluorescence diagnostics of Ce6 distribution in the NIR range and PDT with simultaneous assessment of hemoglobin oxygenation and tumor blood flow. Fluorescence diagnostics and PDT were performed using the single laser λexc=660 ± 5 nm. Results: Combined spectroscopic PDT monitoring method allowed simultaneous estimation of Ce6 photobleaching, hemoglobin oxygenation and tumor vascular thrombosis during PDT without interrupting the therapeutic light exposure. Conclusion: The developed method of tumor phototheranostics using chlorin-type PSs may make it possible to personalize the duration of therapeutic light exposure during PDT.

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]

[Figure: see text].

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.

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]

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]

A spectroscopic study of the photophysical properties of methylene blue (MB) in aqueous solutions was carried out. Absorption and fluorescence spectra as well as fluorescence lifetime were recorded. The concentration dependence of the intensity and shape of the spectra allowed establishing the ranges of MB concentrations for in vitro and in vivo studies at which aggregation is not observed (up to 0.01 mM, which corresponds to 3.2 mg/kg). Studies of photodegradation in biological media showed that photobleaching of more than 80% in plasma and culture media is observed already at a dose of 5 J/cm2 , while in water at this concentration and dose photobleaching is not yet observed, and at a dose of 50 J/cm2 photobleaching of MB is about 30%. It was found that in media containing proteins and having an alkaline pH, photobleaching occurs significantly faster than in neutral aqueous media. The ionic strength of the solution has no effect on the photobleaching rate. Such photobleaching is caused by the photodegradation of MB rather than the transition to the leucoform.The efficiency of singlet oxygen generation and photodynamic activity were evaluated in vitro. In the investigated range of MB concentrations, the efficiency of singlet oxygen generation is rather low, because positively charged MB binds to negatively charged cell membranes, which leads to a change in the type of photodynamic reaction. The emergence of other reactive oxygen species (ROS), different from singlet oxygen, in cells has been demonstrated. The generation of ROS and the low quantum yield of singlet oxygen generation indicate the tendency of MB to provide the type I photosensitization mechanism (electron transfer with the formation of semi-reduced and semi-oxidized MB+ radicals) rather than to the type II mechanism (energy transfer to oxygen with the formation of singlet oxygen) in biological media and in vivo.

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]

Fluorescence interference is a common issue when analyzing microplastics with Raman spectroscopy due to the presence of coloring substances such as organic dyes. Various methods have been attempted to mitigate this fluorescent background, with photobleaching being widely used. However, there has been a lack of quantitative studies on the extent of photobleaching at the single-particle level. In this study, we quantitatively analyzed the photobleaching effect by varying microplastic size, dye concentration, and laser intensity, revealing the magnitude of this effect and its dependence on these parameters. Specifically, we analyzed how quickly fluorescence decreases at the single-particle level, considering the photobleaching efficiency of typical organic dyes. Our results show that, under realistic Raman measurement conditions, careful adjustment of these variables can lead to rapid photobleaching of microplastics below a certain size, thereby sufficiently eliminating fluorescence background issues. These insights are crucial for optimizing experimental protocols and streamlining the detection of small colored microplastics in real environmental samples using Raman spectroscopy. [Display omitted] • Fluorescence interference is a common issue when analyzing microplastics using Raman spectroscopy. • There has been a lack of quantitative studies for photobleaching at the single particle level. • Microplastics less than 5 μm could be detected within 27 s through photobleaching. • The fluorescent background can be minimized due to photobleaching by considering values such as plastic size and laser power. [ABSTRACT FROM AUTHOR]

Severe photobleaching in two-photon excitation fluorescence microscopy (TPM) has limited its potential for long-term and quantitative imaging of live cells and tissues. One solution is to excite fluorophores with a high-repetition rate which reduces the peak intensity of irradiance. However, there is a lack of knowledge about the general utility of this strategy for fluorescent proteins. Here, using simple photobleaching assay, we studied the photobleaching of EGFP and tdTomato at 80 MHz and 640 MHz repetition rates. Our analysis shows that the impact of high repetition rate excitation on reducing photobleaching in TPM is highly dependent on the excitation wavelength and fluorophores. Our results are useful for selecting optimal parameters to minimize photobleaching and photodamage in TPM. • High repetition rate excitation (>80 MHz) helps reduce photobleaching. • The effect of high repetition rate excitation highly depends on the illumination wavelengths as well as fluorophores. • A simple but robust and quantitative assay to measure the photobleaching rates of fluorescent proteins is provided. [ABSTRACT FROM AUTHOR]

[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]

Self-written polymer waveguide is a polymer interconnection structure often integrated with optical fibers. The structure is fabricated by photopolymerization, through which a photon beam initiates a polymerization reaction to change liquid monomer into a solid polymer. During light-matter interaction photoinitiator molecules are excited to the singlet and then to the third state to form free radicals by attaching to the adjacent monomer molecules. The free radicals in turn form polymer chains and cross-linked structures. Photo-induced polymerization leads to a raised refractive index of the monomer and forms an optical waveguide. The change is permanent to allow the process of self-focusing and self-trapping of the propagation light beam and hence guide it to the aligned optical fiber. In this article, light matter interaction during polymer waveguide fabrication and post polymerization has been investigated. A photopolymer system of acrylate-based monomer, co-initiator amine and photo initiator is used to study the absorption of the monomer mixture at the spectral range of 450–550 nm. Then photobleaching was investigated after waveguide fabrication. The optical transmission efficiency of the polymer bridge increases as absorption ceases within the polymer structure such that optical loss reduces from 1.1 dB to about 0.65 dB. [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).