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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]

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., (© 2024 Wiley‐VCH GmbH.)

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

Advanced 3D high-resolution imaging techniques are essential for investigating biological challenges, such as neural circuit analysis and tumor microenvironment in intact tissues. However, the fluorescence signal emitted by endogenous fluorescent proteins in cleared or expanded biological samples gradually diminishes with repeated irradiation and prolonged imaging, compromising its ability to accurately depict the underlying scientific problem. We have developed a strategy to preserve fluorescence in cleared and expanded tissue samples during prolonged high-resolution three-dimensional imaging. We evaluated various compounds at different concentrations to determine their ability to enhance fluorescence intensity and resistance to photobleaching while maintaining the structural integrity of the tissue. Specifically, we investigated the impact of EDTP utilization on GFP, as it has been observed to significantly improve fluorescence intensity, resistance to photobleaching, and maintain fluorescence during extended room temperature storage. This breakthrough will facilitate extended hydrophilic and hydrogel-based clearing and expansion methods for achieving long-term high-resolution 3D imaging of cleared biological tissues by effectively safeguarding fluorescent proteins within the tissue., (© 2024. The Author(s).)

Fluorescence microscopy is limited by photoconversion due to continuous illumination, which results in not only photobleaching but also conversion of fluorescent molecules into species of different spectral properties through photoblueing. Here, we determined different fluorescence parameters of photoconverted products for various fluorophores under standard confocal and stimulated emission depletion (STED) microscopy conditions. We observed changes in both fluorescence spectra and lifetimes that can cause artifacts in quantitative measurements, which can be avoided by using exchangeable dyes., (© 2024. The Author(s).)

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.

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

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

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

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

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

Porphyrin metal-organic frameworks (MOFs) are widely used in photocatalytic advanced oxidation processes (AOP). However, the stability and deactivation of MOFs, crucial for reusability, have been understudied compared to their catalytic activity. We investigated photobleaching in porphyrin MOFs PCN-224-M (M= H 2 , Fe, Co, Cu, Zn) under visible light and H 2 O 2. The MOFs exhibited crystallinity loss, ring-opening cleavage, and linker degradation. Photobleaching resulted from direct redox reactions between porphyrin sites and H 2 O 2. Metal-oxo-porphyrin intermediates played a key role in the "group effect," with different functional groups affecting the photobleaching rate: PCN-224-Fe ≈ PCN-224-Co > PCN-224-H 2 > PCN-224-Cu ≈ PCN-224-Zn. This trend related to chelated metal ions' electronic structures and their propensity for metal-oxo intermediate formation, establishing a structure-stability relationship. Our study enhances understanding of deactivation mechanisms in porphyrin MOFs during AOP, aiding the design of resilient and efficient MOF catalysts for environmental applications. [Display omitted] • Photobleaching deactivations of porphyrin MOF PCN-224-M in photocatalytic AOP scenarios were observed. • The MOFs underwent the loss of crystallinity, ring-opening cleavage and porphyrin degradation in photobleaching deactivation. • The photobleaching mechanism was attributed to direct redox reaction between porphyrin sites and H 2 O 2. • Influence of the chelated metal ions on stability in AOP scenario is revealed. • The structure-stability relationship is established based on the propensity for metal-oxo intermediate formation. [ABSTRACT FROM AUTHOR]

A supramolecular photosensitizer system SQ-DPA⊃CB[8], achieving through host–guest interactions between the NIR squaraine dye SQ-DPA and cuburbit[8]uril (CB[8]), self-assembles into uniform cubes, demonstrating excellent performance in fluorescence imaging and photodynamic therapy of mice bearing HepG2 tumors. [Display omitted] • SQ-DPA⊃CB[8] has the capability for tumor imaging and photodynamic therapy (PDT). • Photosensitizer system selectively enhance accumulation in the mitochondria of tumor cells. • SQ-DPA⊃CB[8] complexes effectively improve ROS generation efficiency (2.6 times enhancement). The development of a multifunctional system capable of selectively performing fluorescence imaging within tumors and exhibiting enhanced anti-tumor efficacy represents a significant advancement in fluorescence imaging-guided tumor treatment. Near-infrared (NIR) fluorescent photosensitizers have emerged as promising tools for tumor diagnosis and therapy due to their ability to minimize background interference and penetrate deeper tissues. However, there remain limitations that impede their practical application. Herein, we propose a supramolecular photosensitizer system, SQ-DPA⊃CB[8], achieved through host–guest interactions between the NIR squaraine dye SQ-DPA and cuburbit[8]uril (CB[8]) for tumor imaging and photodynamic therapy (PDT). Leveraging the excellent recognition and encapsulation properties of CB[8], the photosensitizer system self-assembles into uniform cubes. Compared to the precursor SQ-DPA, the assembled SQ-DPA⊃CB[8] supramolecular complexes selectively enhancing accumulation in the mitochondria of tumor cells, while substantially reduce dark toxicity. Furthermore, they effectively alleviate photobleaching of SQ-DPA, significantly extending the duration of phototherapy. In vitro and in vivo experiments emphasize the enhanced photodynamic therapy potential of SQ-DPA⊃CB[8] complexes. This work presents a novel approach for constructing high-performance imaging-guided photosensitizers through supramolecular self-assembly, addressing the limitations encountered in current applications. [ABSTRACT FROM AUTHOR]

Coumarin Based Glyoxylate Photoinitiators for Free Radical and Cationic Photopolymerizations with UV–Visible LED Irradiation. The novel photoinitiators coumarin-3-oxoacetic acids methyl esters (COAEs) exhibited versatile talents working as photoinitiators in photopolymerization. Upon absorption of ultraviolet (UV) or visible (Vis) light, COAEs would generate active free redicals via Type I photoinitiators mechanism or capture hydrogen via Type II photoinitiators mechanism to initiate free radical polymerization (FRP) of acrylic esters Tri-(propylene glycol) diacrylate (TPGDA). Moreover, they can sensitize sulfonium salts and iodonium salts to to initiate cationic photopolymerization (CP) of epoxy monomers and oxetane monomers, respectively. Additionally, they can initiate the deep curing of TPGDA to a depth of 9cm at extremely low concentrations of COAEs (0.01 wt%) with a 415 nm and 450 nm LED (100 mW · cm−2) in acceptable exposure time (5 min). The parent nucleus of COAEs is coumarin, which can be extracted from plants such as strawberries, tonka bean, blackcurrant, etc., thus COAEs exhibit excellent cell compatibility. [Display omitted] • Coumarin-3-oxoacetic acid methyl esters (COAEs) were synthesized as photoinitiators suitable for UV–Vis LED irradiation. • COAEs could sensitize iodonium salts and sulfonium salts to initiate cationic photopolymerization. • COAEs can initiate deep curing of acrylate monomer to approach to 9 cm depth at acceptable low concentrations (0.01 wt%). • COAEs exhibit good photobleaching and cell compatibility. High-performance photoinitiators (PIs) play a crucial role in ultraviolet–visible (UV–Vis) photopolymerization. In this study, three coumarin-3-oxoacetic acid methyl esters (COAEs) which demonstrate significant UV–Vis absorption within the 300–500 nm range were synthesized and studied. Real-time fourier transform infrared (RT-FTIR) and photo-differential scanning calorimetry (photo-DSC) experiments demonstrated that these PIs can function as Type I and Type II PIs for initiating free radical polymerization (FRP) of acrylate monomers. Additionally, they can serve as photosensitizers for iodonium salts and sulfonium salts, enabling cationic photopolymerization (CP) of epoxy and oxetane monomers. Remarkably, even at low concentrations of 0.01 wt%, they can effectively initiate the deep curing of Tri-(propylene glycol) diacrylate (TPGDA) to a depth of approximately 9 cm after 5 min of irradiation with a 415 nm or 450 nm LED light source. Furthermore, the COAEs exhibit satisfactory photobleaching properties, making them suitable for colorless applications in photopolymerization. The photochemical and photophysical properties of the COAEs were thoroughly investigated using absorption spectroscopy, electron spin resonance, cyclic voltammetry experiments, and photoinduced decarboxylation experiments to elucidate the photoinitiation mechanism. Cytotoxicity experiments revealed that COAE-N exhibited good cytocompatibility. Therefore, the exceptional polymerization properties of COAEs offer promising prospects for FRP and CP applications. Additionally, their efficient photobleaching characteristics make them highly suitable for deep photopolymerization applications. Moreover, the low cytotoxicity of COAEs suggests significant potential for biomedical applications such as dental restoration. [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.

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

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

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

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

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

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

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

We present a polymer thermo-optic mode switch based on the structure of two vertical Mach-Zehnder interferometers (MZIs). It can achieve a rapid switch between E00, E01, E10 and E11 with the different heaters turning on. In our simulation, we compare two structures (the metal top electrode and the graphene electrode). The Finite element method (FEM) is used to simulate the parameters of the graphene electrode device. The power consumption of 1 and 3 heaters is 1.72 mW, and that of 2 and 4 heaters is 1.85 mW. In addition, the response speeds of the 1 and 3 graphene heater mode switch calculated are 36.9 μs (rise) and 103.4 μs (down), and that of 2 and 4 graphene heater mode switch calculated are 17.3 μs (rise) and 87.9 μs (down). The proposed model can be used in the field of low power consumption mode division and multiplexer of three-dimensional integrated.

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

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

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.

The oligomerization of proteins is an important biological control mechanism and has several functions in activity and stability of enzymes, structural proteins, ion channels and transcription factors. The determination of the relevant oligomeric states in terms of geometry (spatial extent), oligomer size (monomer or dimer or oligomer) and affinity (amounts of monomer, dimer and oligomer) is a challenging biophysical problem. Förster resonance energy transfer and fluorescence fluctuation spectroscopy are powerful tools that are sensitive to proximity and oligomerization respectively. Here it is proposed to combine image-based lifetime-detected Forster resonance energy transfer with image correlation spectroscopy and photobleaching to determine distances, oligomer sizes and oligomer distributions. Simulations for simple oligomeric forms illustrate the potential to improve the discrimination between different quaternary states in the cellular milieu., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author. Published by Elsevier B.V. All rights reserved.)

Here we present a method to reduce the photobleaching of fluorescent proteins and the associated phototoxicity. It exploits a photophysical process known as reverse intersystem crossing, which we induce by near-infrared co-illumination during fluorophore excitation. This dual illumination method reduces photobleaching effects 1.5-9.2-fold, can be easily implemented on commercial microscopes and is effective in eukaryotic and prokaryotic cells with a wide range of fluorescent proteins., (© 2023. The Author(s).)

Optical tissue clearing and three-dimensional (3D) immunofluorescence (IF) microscopy is transforming imaging of the complex tumor microenvironment (TME). However, current 3D IF microscopy has restricted multiplexity; only 3 or 4 cellular and noncellular TME components can be localized in cleared tumor tissue. Here we report a light-emitting diode (LED) photobleaching method and its application for 3D multiplexed optical mapping of the TME. We built a high-power LED light irradiation device and temperature-controlled chamber for completely bleaching fluorescent signals throughout optically cleared tumor tissues without compromise of tissue and protein antigen integrity. With newly developed tissue mounting and selected region-tracking methods, we established a cyclic workflow involving IF staining, tissue clearing, 3D confocal microscopy, and LED photobleaching. By registering microscope channel images generated through 3 work cycles, we produced 8-plex image data from individual 400 μm-thick tumor macrosections that visualize various vascular, immune, and cancer cells in the same TME at tissue-wide and cellular levels in 3D. Our method was also validated for quantitative 3D spatial analysis of cellular remodeling in the TME after immunotherapy. These results demonstrate that our LED photobleaching system and its workflow offer a novel approach to increase the multiplexing power of 3D IF microscopy for studying tumor heterogeneity and response to therapy., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Significance: Light-sheet fluorescence microscopy (LSFM) has emerged as a powerful and versatile imaging technique renowned for its remarkable features, including high-speed 3D tomography, minimal photobleaching, and low phototoxicity. The interference light-sheet fluorescence microscope, with its larger field of view (FOV) and more uniform axial resolution, possesses significant potential for a wide range of applications in biology and medicine., Aim: The aim of this study is to investigate the interference behavior among multiple light sheets (LSs) in LSFM and optimize the FOV and resolution of the light-sheet fluorescence microscope., Approach: We conducted a detailed investigation of the interference effects among LSs through theoretical derivation and numerical simulations, aiming to find optimal parameters. Subsequently, we constructed a customized system of multi-LSFM that incorporates both interference light sheets (ILS) and noninterference light-sheet configurations. We performed beam imaging and microsphere imaging tests to evaluate the FOV and axial resolution of these systems., Results: Using our custom-designed light-sheet fluorescence microscope, we captured the intensity distribution profiles of both interference and noninterference light sheets (NILS). Additionally, we conducted imaging tests on microspheres to assess their imaging outcomes. The ILS not only exhibits a larger FOV compared to the NILS but also demonstrates a more uniform axial resolution., Conclusions: By effectively modulating the interference among multiple LSs, it is possible to optimize the intensity distribution of the LSs, expand the FOV, and achieve a more uniform axial resolution., (© 2024 The Authors.)

Fluorescent dyes synergize with advanced microscopy for researchers to investigate the location and dynamic processes of biomacromolecules with high spatial and temporal resolution. However, the instability of fluorescent dyes, including photobleaching and photoconversion, represent fundamental limits for super-resolution and time-lapse imaging. In this review, we discuss the latest advances in improving the photostability of fluorescent dyes. We summarize the primary photobleaching processes of cyanine and rhodamine dyes and highlight a range of strategies developed in recent years to strengthen these fluorophores. Additionally, we discuss the influence of protein microenvironments and labeling methods on the photostability of fluorophores. We aim to inspire next-generation robust and bright fluorophores that ultimately enable the routine practice of time-lapse super-resolution imaging of live cells., Competing Interests: Declaration of competing interest Z.C. is an inventor of a patent on mitochondria dyes described in this review (CN 202010492298.8). Z.C. and Y.Z. are the inventors of a patent on the fluorophores with S-arylation-type conjugation chemistry in this review (CN202210228995.1, patent pending)., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Context.—: Photobleaching artifact occurs when fluorescence intensity decreases following light exposure. Slides stained with fluorescent techniques may be stored in the dark until primary diagnostics. Experimental evidence suggesting the rate of photobleaching and necessity of dark storage is lacking., Objective.—: To compare photobleaching rate on direct immunofluorescence and Thioflavin T slides stored in ambient room light conditions and exposed to excitatory wavelengths., Design.—: During 2 iterations of the experiment, 45 slides were prepared, 42 with immunofluorescent antibodies plus 3 with thioflavin, from skin and kidney biopsies. The experimental group was stored in room light conditions in comparison to the control in the dark, at room temperature. Further, 1 immunofluorescence slide and 1 thioflavin slide were exposed to excitatory fluorescent light for several hours. Significant photobleaching was defined as an integer decrease in score (scale, 0-3)., Results.—: Exposure times ranged from 152 to 3034 hours. Nine of the 42 immunofluorescence slides (21%) photobleached after a minimum exposure of 152 hours to room light, with no significant difference between the experimental and control groups (all P values >.05). The immunofluorescence slide exposed to fluorescent light for 4 hours showed marked photobleaching in the exposed field but not elsewhere. No thioflavin slides showed clinically significant photobleaching under any conditions., Conclusions.—: Clinically significant photobleaching of slides exposed to room light may occur after a few days, but not a few hours (unless exposed to excitatory fluorescent light). Conversely, thioflavin-stained slides did not photobleach when exposed to ambient room air and photobleached only negligibly when exposed to excitatory fluorescent light., Competing Interests: The authors have no relevant financial interest in the products or companies described in this article., (© 2024 College of American Pathologists.)

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

• Method enables investigation of amyloid-β oligomer stoichiometry without requiring extrinsic fluorescent probes. • Uses single-molecule stepwise photobleaching in vitro. • Unveils heterogeneity within populations of oligomers. • Assays oligomer-induced dysregulation of intracellular Ca2+ homeostasis in living cells. A major hallmark of Alzheimer's disease is the misfolding and aggregation of the amyloid- β peptide (Aβ). While early research pointed towards large fibrillar- and plaque-like aggregates as being the most toxic species, recent evidence now implicates small soluble Aβ oligomers as being orders of magnitude more harmful. Techniques capable of characterizing oligomer stoichiometry and assembly are thus critical for a deeper understanding of the earliest stages of neurodegeneration and for rationally testing next-generation oligomer inhibitors. While the fluorescence response of extrinsic fluorescent probes such as Thioflavin-T have become workhorse tools for characterizing large Aβ aggregates in solution, it is widely accepted that these methods suffer from many important drawbacks, including an insensitivity to oligomeric species. Here, we integrate several biophysics techniques to gain new insight into oligomer formation at the single-molecule level. We showcase single-molecule stepwise photobleaching of fluorescent dye molecules as a powerful method to bypass many of the traditional limitations, and provide a step-by-step guide to implementing the technique in vitro. By collecting fluorescence emission from single Aβ(1–42) peptides labelled at the N-terminal position with HiLyte Fluor 555 via wide-field total internal reflection fluorescence (TIRF) imaging, we demonstrate how to characterize the number of peptides per single immobile oligomer and reveal heterogeneity within sample populations. Importantly, fluorescence emerging from Aβ oligomers cannot be easily investigated using diffraction-limited optical microscopy tools. To assay oligomer activity, we also demonstrate the implementation of another biophysical method involving the ratiometric imaging of Fura-2-AM loaded cells which quantifies the rate of oligomer-induced dysregulation of intracellular Ca2+ homeostasis. We anticipate that the integrated single-molecule biophysics approaches highlighted here will develop further and in principle may be extended to the investigation of other protein aggregation systems under controlled experimental conditions. [ABSTRACT FROM AUTHOR]

We investigated the radiation-induced attenuation (RIA) at 1550 nm under X-rays in two single-mode germanosilicate optical fibers (OFs): the Corning SMF-28e+ and a homemade fiber manufactured by the Université Côte d’Azur in the framework of the CERTYF project. The RIA dependence on the injected power level of the probing signal is studied in order to analyze the photobleaching phenomenon at high doses [>100 kGy(SiO2)]. The Corning SMF-28e+ revealed no RIA variation with the signal power (microwatts–milliwatts range), proving to be a good candidate for distributed OF sensor (DOFS) applications, while the CERTYF OF showed a clear photobleaching effect. A fitting routine based on the second-order growth kinetics model of Griscom has been performed, which has shown good consistency with the experimental results and offered a quantitative evaluation of the photobleaching effect on the CERTYF OF. [ABSTRACT FROM AUTHOR]

We examined the effect of Bradyrhizobium japonicum FeCh (BjFeCh) expression on the regulation of porphyrin biosynthesis and resistance to norflurazon (NF)-induced photobleaching in transgenic rice. In response to NF, transgenic lines F4 and F7 showed lesser declines in chlorophyll, carotenoid, Fv/Fm, ϕPSII, and light-harvesting chlorophyll (Lhc) a/b-binding proteins as compared to wild-type (WT) plants, resulting in the alleviation of NF-induced photobleaching. During photobleaching, levels of heme, protoporphyrin IX (Proto IX), Mg-Proto IX (monomethylester), and protochlorophyllide decreased in WT and transgenic plants, with lesser decreases in transgenic plants. Most porphyrin biosynthetic genes were greatly downregulated in WT and transgenic plants following NF treatment, with higher transcript levels in transgenic plants. The expression of BjFeCh in transgenic rice may play a protective role in mitigating NF-induced photobleaching by maintaining levels of heme, chlorophyll intermediates, and Lhc proteins. This finding will contribute to understanding the resistance mechanism of NF-resistant crops and establishing a new strategy for weed control. [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.

Stress fibers (SFs) in cells transmit external forces to cell nuclei, altering the DNA structure, gene expression, and cell activity. To determine whether SFs are involved in mechanosignal transduction upon intraluminal pressure, this study investigated the SF direction in smooth muscle cells (SMCs) in aortic tissue and strain in the SF direction. Aortic tissues were fixed under physiological pressure of 120 mmHg. First, we observed fluorescently labeled SFs using two-photon microscopy. It was revealed that SFs in the same smooth muscle layers were aligned in almost the same direction, and the absolute value of the alignment angle from the circumferential direction was 16.8° ± 5.2° (n = 96, mean ± SD). Second, we quantified the strain field in the aortic tissue in reference to photo-bleached markers. It was found in the radial-circumferential plane that the largest strain direction was − 21.3° ± 11.1°, and the zero normal strain direction was 28.1° ± 10.2°. Thus, the SFs in aortic SMCs were not in line with neither the largest strain direction nor the zero strain direction, although their orientation was relatively close to the zero strain direction. These results suggest that SFs in aortic SMCs undergo stretch, but not maximal and transmit the force to nuclei under intraluminal pressure. [ABSTRACT FROM AUTHOR]

Important breakthroughs in far-field imaging techniques have been made since the first demonstrations of stimulated emission depletion (STED) microscopy. To date, the most straightforward and widespread deployment of STED microscopy has used continuous wave (CW) laser beams for both the excitation and depletion of fluorescence emission. A major drawback of the CW STED imaging technique has been photobleaching effects due to the high optical power needed in the depletion beam to reach sub-diffraction resolution. To overcome this hurdle, we have applied a synchronous detection approach based on modulating the excitation laser beam, while keeping the depletion beam at CW operation, and frequency filtering the collected signal with a lock-in amplifier to record solely the super-resolved fluorescence emission. We demonstrate here that such approach allows an important reduction in the optical power of both laser beams that leads to measurable decreases in photobleaching effects in STED microscopy. We report super-resolution images with relatively low powers for both the excitation and depletion beams. In addition, typical unwanted scattering effects and background signal generated from the depletion beam, which invariably arises from mismatches in refractive index in the material composing the sample, are largely reduced by using the modulated STED approach. The capability of acquiring super-resolution images with relatively low power is quite relevant for studying a variety of samples, but particularly important for biological species as exemplified in this work. [ABSTRACT FROM AUTHOR]

mKate red-to-green photoconversion is a non-canonical type of phototransformation in fluorescent proteins, with a poorly understood mechanism. We have hypothesized that the daughter mKate2 protein may also be photoconvertible, and that this phenomenon would be connected with mKate(2) chromophore photoreduction. Indeed, upon the intense irradiation of the protein sample supplemented by sodium dithionite, the accumulation of green as well as blue spectral forms is enhanced. The reaction was shown to be reversible upon the reductant's removal. However, an analysis of the fluorescence microscopy data, absorption spectra, kinetics and time-resolved fluorescence spectroscopy revealed that the short-wavelength spectral forms of mKate(2) exist before photoactivation, that their fractions increase light-independently after dithionite addition, and that the conversion is facilitated by the photobleaching of the red chromophore form. [ABSTRACT FROM AUTHOR]