A Molecular Logic Gate Enables Single-Molecule Imaging and Tracking of Lipids in Intracellular Domains.

Photoactivatable dyes enable single-molecule imaging and tracking in biology. Despite progress in the development of new fluorophores and labeling strategies, many intracellular compartments remain difficult to image beyond the limit of diffraction in living cells. For example, lipid domains, e.g., membranes and droplets, remain difficult to image with nanometric resolution. To visualize these challenging subcellular targets, it is necessary to develop new fluorescent molecular devices beyond simple on/off switches. Here, we report a fluorogenic molecular logic gate that can be used to image single molecules associated with lipid domains, most notably droplets, with excellent specificity. This probe requires the subsequent action of light, a lipophilic environment, and a competent nucleophile to produce a fluorescent product. The combination of these inputs results in a probe that can be used to image the boundary of lipid droplets in three dimensions with resolution beyond the limit of diffraction. Moreover, this probe enables single-molecule tracking of lipid trafficking between droplets and the endoplasmic reticulum.

Reversible spatial and temporal control of lipid signaling.

Herein, we introduce versatile molecular tools that enable specific delivery and visualization of photoswitchable lipids at cellular membranes, namely at the plasma membrane and internal membranes. These molecules were prepared by tethering ortho-nitrobenzyl-based fluorescent cages with a signaling lipid bearing an azobenzene photoswitch. They permit two sequential photocontrolled reactions, which are uncaging of a lipid analogue and then its repeated activation and deactivation. We used these molecules to activate GPR40 receptor transiently expressed in HeLa cells and demonstrated downstream modulation of intracellular Ca2+ levels.

Gas-phase Förster resonance energy transfer in mass-selected ions with methylene or peptide linkers between two dyes: a concerted dance of charges.

Förster Resonance Energy Transfer (FRET) between a photoexcited and a ground-state dye is dictated by how far apart the two dyes are compared to the Förster distance. While there is a significant number of studies on the process for biomacromolecules in solution, there are only a few reports on gas-phase FRET. Here we report on a simple gas-phase model system, synthesized with the rhodamine 575 (R575+) and rhodamine 640 (R640+) FRET pair and a covalent linker with four methylenes, R575+-(CH2)4-R640+. Each dye carries a positive charge which allows for mass-spectroscopy experiments. We have recorded gas-phase dispersed fluorescence spectra of the mass-selected dications excited at different wavelengths using the homebuilt LUNA (LUminescence iNstrument in Aarhus) setup and find in all cases that emission is exclusively from the R640+ acceptor dye. The linker does not interfere electronically with the dyes and simply acts as a spacer. We can therefore establish the direct effect of the interaction between the two dyes when it comes to emission band maximum. Indeed, we find that R640+ experiences a significant shift in its maximum from 560 ± 1 nm for the monomer cation to 577 ± 2 nm in the presence of R575+, independent of initial excitation of R575+ or R640+. This redshift is ascribed to the large polarizability along the long axis of the xanthene core structure, and that this polarizability is larger in the excited state than in the ground state. Experiments were also done on a triply charged 11-mer peptide labelled with the same two dyes, R575+-(Gly-Gln)5-Lys-R640+ + H+ (Gly = glycine, Gln = glutamine, and Lys = lysine) where the extra positive charge is located on the peptide. Again a redshifted emission spectrum of the donor is observed with maximum at 582 ± 2 nm. Our work clearly demonstrates strong sensitivity of the photophysics of one dye to the nearby environment, and that caution is needed when using the energy transfer efficiency to infer dye-dye separations in gas-phase experiments.

Microscope laser assisted photooxidative activation of bioorthogonal ClickOx probes.

A photoactivatable fluorogenic tetrazine-rhodaphenothiazine probe was synthesized and studied in light-assisted, bioorthogonal labeling schemes. Experimental results revealed that the bioorthogonally conjugated probe efficiently sensitizes 1O2 generation upon illumination with green or orange light and undergoes self-oxidation leading to an intensely fluorescent sulfoxide product. An added value of the present probe is that it is also suitable for STED super-resolution microscopy using a 660 nm depletion laser.

Enhancing the performance of pollution degradation through secondary self-assembled composite supramolecular heterojunction photocatalyst BiOCl/PDI under visible light irradiation.

A novel n-n type inorganic/organic heterojunction of flaky-like BiOCl/PDI photocatalyst was constructed by water bath heating method. Meanwhile, a simple method - secondary self-assembly was used to prepare the BiOCl/PDI with a special band structure. The photocatalytic activities were evaluated by degrading aqueous organic pollutants under visible light (λ > 420 nm). The removal rates of 5 mg L-1 phenol (non-ionic type), methyl orange (MO, anionic type), rhodamine B (RhB, cationic type) and 10 mg L-1 RhB by secondary self-assembly BiOCl/PDI (BiOCl/PDI-2) were 8.0%, 3.4%, 27.8% and 78.9% higher than self-assembly BiOCl/PDI (BiOCl/PDI-1) under visible light (λ > 420 nm). The better photocatalytic activity for BiOCl/PDI-2 was attributed to the optimization of energy-band structures, which arose from different exposed surfaces, narrower interplanar spacing and stronger visible light absorption performance. Under acidic condition, BiOCl/PDI-2 showed a good photocatalytic activity, which was not affected by neutral ionic intensity and had good recycling properties. Moreover, the photocatalytic mechanism was explored by free radical capture test and electron paramagnetic resonance (EPR), and contribution of active species was calculated. The main active species of BiOCl/PDI-2 were ·O2-, 1O2 and h+. Our work may provide a route to design efficient inorganic/organic heterojunctions for organic pollutants degradation.

Fluorimetric-Based Method to Detect and Quantify Total S-Nitrosothiols (SNOs) in Plant Samples.

Accumulating experimental evidence indicates that S-nitrosylation (technically S-nitrosation) events have a central role in plant biology, presumably accounting for much of the widespread influence of nitric oxide (NO) on developmental, metabolic, and stress-related plant responses. Therefore, the accurate detection and quantification of S-nitrosylated proteins and peptides can be particularly useful to determine the relevance of this class of compounds in the ever-increasing number of NO-dependent signaling events described in plant systems. Up to now, the quantification of S-nitrosothiols (SNOs) in plant samples has mostly relied on the Saville reaction and the ozone-based chemiluminescence method, which lacks sensitivity and are very time-consuming, respectively. Taking advantage of the photolytic properties of S-nitrosylated proteins and peptides, the method described in this chapter allows simple, fast, and high-throughput detection of SNOs in plant samples.

Role of Fe(III) in aqueous solution or deposited on ZnO surface in the photoassisted degradation of rhodamine B and caffeine.

Iron (III) was incorporated, to the surface of a synthesized ZnO, using two nominal molar percentages of Fe (III): 1% and 5% Fe relative to ZnO. Samples dried and calcined at 200 °C and 400 °C for 2 h, were characterized by XRD, XPS, XRF, N2-adsorption-BET and (UV-vis)-DRS. Photocatalytic activities of the catalysts were assessed based on the degradation of rhodamine B (RhB) and caffeine (CAF) in aqueous solution under two irradiation conditions: UV and visible light illumination. Prior to the photocatalytic tests, the interaction of each one of the substrates with either Fe(III) or Fe(II) was studied in homogeneous medium under UV-illumination and oxygenated environment. It was found that Fe (III) can play an important role in homogeneous media in the photoassisted degradation, both of rhodamine B and caffeine, while Fe (II) does not exert a relevant role in the photoassisted degradation of the referred substrates. Fe-ZnO samples display similar or poorer performance than pure ZnO in the presence of UV light for both studied substrates. The phenomenon can be attributed to the formation of either goethite or ZnFe2O4 at the ZnO surface where the coupled Fe3+/Fe2+ can act as recombination centers for the photogenerated charges. On the contrary, all Fe-ZnO samples showed enhanced photocatalytic activity under visible illumination which seems to be independent of the iron content. In this context, the mechanisms for photoassisted degradation of both the substrates in homogeneous medium and photocatalytic degradation are discussed, as well as the role of Fe in the photodegradation processes.

Integration of plasmonic effect into MIL-125-NH2: An ultra-efficient photocatalyst for simultaneous removal of ternary system pollutants.

Industrial effluents often contain mixed metal ions and dyes, and it is difficult to efficiently remove both types of contaminants simultaneously. Here, MIL-125-NH2@Ag/AgCl composites were for the first time developed through a facile deposition-photoreduction method for simultaneously removing Cr(VI)/Rhodamine B (RhB)/Malachite Green (MG) ternary system pollutants under visible-light irradiation. The capacities of Cr(VI) reduction dramatically increased to 98.4% in the coexistence of RhB and MG compared to that of binary (Cr(VI)/RhB (69.6%) or Cr(VI)/MG (67.5%)) and single Cr(VI) (29%) systems. In the meantime, the degradation efficiencies of dyes especially RhB in the ternary system were also improved compared to that of their individual systems. On the grounds of all the experimental results, it can be concluded that the efficient light-harvesting and electrons migration in MIL-125-NH2@Ag/AgCl and the synergistic effect of redox reactions between Cr(VI) and dyes hinder the recombination of photo-induced electron-hole pairs, which are responsible for their high photocatalytic activity to eliminate the mixed pollutants. This study provides a new route to construct high-performance photocatalysts for the practical treatment of wastewater containing mixed pollutants.

Photoactivation of silicon rhodamines via a light-induced protonation.

Photoactivatable fluorophores are important for single-particle tracking and super-resolution microscopy. Here we present a photoactivatable fluorophore that forms a bright silicon rhodamine derivative through a light-dependent protonation. In contrast to other photoactivatable fluorophores, no caging groups are required, nor are there any undesired side-products released. Using this photoactivatable fluorophore, we create probes for HaloTag and actin for live-cell single-molecule localization microscopy and single-particle tracking experiments. The unusual mechanism of photoactivation and the fluorophore's outstanding spectroscopic properties make it a powerful tool for live-cell super-resolution microscopy.

Design and Synthesis of an Activatable Photoacoustic Probe for Hypochlorous Acid.

Photoacoustic (PA) imaging is a novel imaging modality that combines the high contrast of optical imaging and the deep tissue penetration of ultrasound. PA imaging contrast agents targeting various biological phenomena have been reported, but the development of activatable PA probes, which show a PA signal only in the presence of target molecules, remains challenging in spite of their potential usefulness for real-time PA imaging of specific biomolecules in vivo. To establish a simple design strategy for activatable PA probes, we first designed and synthesized a silicon-rhodamine based near-infrared nonfluorescent dye, wsSiNQ660 (water-soluble SiNQ660), as a scaffold and demonstrated that it offers a high conversion efficiency from light to ultrasound compared to typical near-infrared fluorescent dyes. Importantly, absorption off/on strategies previously established for rhodamine-based fluorescent probes are also applicable to this nonfluorescent dye scaffold. We validated this approach by synthesizing an activatable PA probe for hypochlorous acid (HOCl) and confirmed that it enables three-dimensional imaging of HOCl in mouse subcutis.

Fluorescence imaging of stained red blood cells with simultaneous resonance Raman photostability analysis.

Optical spectroscopic imaging of biological systems has important applications in medical diagnosis, biochemistry, and image-guided surgery. Vibrational spectroscopy, such as Raman scattering, provides high chemical selectivity but is limited by weak signals and a large fluorescence background. Fluorescence imaging is often used by introducing specific dyes in biological systems to label different system parts and to increase the image contrast. However, the extrinsic fluorescence of the staining molecules often masks the intrinsic vibrational signals of biomolecules, which could also be simultaneously detected using the same excitation laser source. Therefore, fluorescence staining is often accompanied by the loss of other important complimentary information. For example, the high laser power often used for the rapid, high-quality imaging could lead to photo-induced suppression or bleaching of the fluorescence and Raman signals resulting in sample photodamage. Therefore, simultaneous imaging and photodamage analysis need to be performed in a controlled bioimaging experiment. Here we perform simultaneous spectroscopic bioimaging and photostability analysis of rhodamine 6G (R6G) stained red blood cells (RBCs) using both fluorescence and resonance Raman imaging in a single 532 nm laser excitation experiment. We develop a corresponding data processing algorithm which allows separation of the two spectroscopic signals. We control the relative intensity of the R6G and RBC signals by varying the excitation laser power and simultaneously monitor the photostability of RBCs. We observe no significant photodamage of RBCs through the absence of changes in the relative Raman peak intensities. Conversely, the R6G molecules show bleaching with the suppression of both the fluorescence and resonance Raman signals. Our approach may be generalized to other types of stained cells with the appropriate selection of fluorescent dyes and excitation sources.

In-situ mineralized robust polysiloxane-Ag@ZnO on cotton for enhanced photocatalytic and antibacterial activities.

A bifunctional interfacial layer was introduced onto the surface of cotton fabric which not only enhanced the interfacial bonding between Ag@ZnO and organic substrates but also improved the photocatalytic performance simultaneously. In detail, a modified cotton fabric (denoted as Cot-g-Si/Ag@ZnO) was fabricated through radiation-induced graft polymerization of γ-methacryloxypropyl trimethoxysilane and followed the in-situ formation of ZnO and loading of Ag nanoparticles simultaneously. Owing to ZnOSi between the graft chains and Ag@ZnO photocatalyst, the charge carrier concentration increased and Ag was prevented from oxidizing through the partial separation from ZnO, leading to enhanced near-field amplitudes of the localized surface plasmon resonance. Cot-g-Si/Ag@ZnO also exhibited excellent photocorrosion resistance, photostability and laundering durability. Its photocatalytic activity was fully maintained after several photodegradation cycles; moreover, after laundering durability test, the photocatalytic activity was improved compared with the newly prepared one. Credible mechanism for the photocatalytic activity of Cot-g-Si/Ag@ZnO under sunlight irradiation is proposed.

Sonophotocatalytic treatment of rhodamine B using visible-light-driven CeO2/Ag2CrO4 composite in a batch mode based on ribbon-like CeO2 nanofibers via electrospinning.

CeO2/Ag2CrO4 composite photocatalyst was successfully fabricated using electrospinning and calcination and chemical precipitation method based on CeO2 ribbon-like fibers and characterized by field-emission scanning electron microscopy (FESEM), energy dispersive X-ray (EDX), X-ray diffraction (XRD), UV-Vis diffuse reflectance spectroscopy (DRS) and Fourier-transform infrared spectroscopy (FT-IR). The as-obtained CeO2/Ag2CrO4 composite used photocatalytic performance in the sonophotodegradation of rhodamine B in aqueous solution under visible-light (LED) irradiation. DRS analysis illustrates that CeO2/Ag2CrO4 composite exhibited enhanced absorption in the visible region-attributed CeO2 nanofibers. The effect of four effective parameters including initial concentration of rhodamine B (RhB), photocatalyst dosage, pH, and irradiation time was studied and optimized using central composite design. The kinetic studies confirmed ability of pseudo first-order reaction based on the Langmuir-Hinshelwood model for fitting empirical data, while its rate constant (kobs), L-H rate constants (kr), and L-H adsorption constants (KA) were 0.0449 min-1, 11.66 mg L-1 min-1 and 1.09E-3 mg L-1, respectively. The enhanced photocatalytic activity could be ascribed to the ultrasound field and formation of a heterojunction system among CeO2 and Ag2CrO4, which lead to a better mass transfer and higher efficiency of charge electron-hole separation, respectively.

Organic Fluorophore Coated Polycrystalline Ceramic LSO:Ce Scintillators for X-ray Bioimaging.

The current effort demonstrates that lutetium oxyorthosilicate doped with 1-10% cerium (Lu2SiO5:Ce, LSO:Ce) radioluminescent particles can be coated with a single dye or multiple dyes and generate an effective energy transfer between the core and dye(s) when excited via X-rays. LSO:Ce particles were surface modified with an alkyne modified naphthalimide (6-piperidin-1-yl-2-prop-2-yn-1-yl-1 H-benzo[ de]isoquinoline-1,3-(2 H)-dione, AlNap) and alkyne modified rhodamine B ( N-(6-diethylamino)-9-{2-[(prop-2-yn-1-yloxy)carbonyl]phenyl}-3 H-xanthen-3-ylidene)- N-ethylethanaminium, AlRhod) derivatives to tune the X-ray excited optical luminescence from blue to green to red using Förster Resonance Energy Transfer (FRET). As X-rays penetrate tissue much more effectively than UV/visible light, the fluorophore modified phosphors may have applications as bioimaging agents. To that end, the phosphors were incubated with rat cortical neurons and imaged after 24 h. The LSO:Ce surface modified with AlNap was able to be successfully imaged in vitro with a low-output X-ray tube. To use the LSO:Ce fluorophore modified particles as imaging agents, they must not induce cytotoxicity. Neither LSO:Ce nor LSO:Ce modified with AlNap showed any cytotoxicity toward normal human dermal fibroblast cells or mouse cortical neurons, respectively.

High-depth fluorescence imaging using a two-photon FRET system for mitochondrial pH in live cells and tissues.

We developed a fluorescent pH probe (1) capable of two-photon excitation and far-visible-emission based on FRET, composed of naphthalimide-piperazine-rhodamine. It exhibited a pH-dependent reversible and fast ratiometric fluorescence change in the rhodamine emission. Probe 1 was applied to image the pH perturbations of mitochondria in living cells and tissues.

Ultra-Bright Rhodamines with Sulfobutylether-ß-Cyclodextrin: A Viable Supramolecular Dye Laser in Aqueous Medium.

Although aqueous dye lasers are much sought after, they have been of no practical use, as laser dyes show a strong tendency for aggregation in water, thus diminishing their optical output. Contributing towards this shortcoming, we studied the noncovalent interactions of two prominent laser dyes, namely, rhodamine 6G and rhodamine B, with a water soluble macrocyclic host, sulfobutylether-ß-cyclodextrin (SBE7 ßCD). Spectral changes in the absorption and fluorescence behavior of dyes in presence of the SBE7 ßCD host indicated adequate complex formation between dye and host (K∼104  M-1 ). A combination of various photophysical parameters evaluated from measurements such as Job plot, changes in the fluorescence lifetime/anisotropy values, and favorable thermodynamic parameters from isothermal titration calorimetric measurements adjudicated a 1 : 1 stoichiometric complex formation between dye and SBE7 ßCD host. Consequently, SBE7 ßCD prevents dye aggregation/adsorption and present rhodamine dyes in their monomeric forms with enhanced fluorescence yield and brightness. These vital parameters were utilized to optimize and demonstrate cost-effective supramolecular broad-band and narrow-band aqueous dye laser systems with improved lasing efficiencies (∼25 % higher for the SBE7 ßCD : RhB system and ∼10 % higher for SBE7 ßCD : Rh6G system), better beam profile, and enhanced durability compared to the respective dyes in optically matched ethanol solutions.

Assessment of the Content of Fluorescent Tracer in Granular Feed Mixture.

Background: This paper describes the use of fluorescence induced by UV radiation to evaluate the share of tracer in feed mixture. Methods: For the purpose of this study, three substances were used. They are as follows: Tinopal, Rhodamine B, and Uranine. Tracer in the form of maize or kardi was added to chicken feed before the mixing process. Grains used in the process were grinded in the mill sieve with a mesh size of 4 and 6 mm. The drawn samples of the mixture were illuminated with UV radiation to make grain tracer light, and then the photo was taken with a digital camera. The acquired images were analyzed with the use of a computer program running on the RGB color model, which was the way to obtain essential information about the percentage share of tracer. Results: It was observed that, in the case of kardi grains, the proposed method gives results significantly deviating from the verification method. Conclusions: Only the tests with the use of maize having an average particle diameter of 2.4 mm and tinted with the solution of Rhodamine B led to acceptable results (consensual with the predetermined verification level).

A novel three-fluorophore system as a ratiometric sensor for multiple protease detection.

A synthetic three-fluorophore system with two enzymatically cleavable linkers has been developed for the simultaneous detection of two proteases in a mixture. The probe was designed to afford single excitation/triple emission ratiometric detection through a fluorescence change during the cleavage of a peptide linker. The developed assays were verified for trypsin and chymotrypsin as the model enzymes.

Study on the photocatalytic reaction kinetics in a TiO2 nanoparticles coated microreactor integrated microfluidics device.

For study of the photocatalytic reaction kinetics in a confined microsystem, a photocatalysis microreactor integrated on a microfluidic device has been fabricated using an on-line UV/vis detector. The performance of the photocatalysis microreactor is evaluated by the photocatalytic degradation of Rhodamine B chosen as model target by using commercial titanium dioxide (Degussa P25, TiO2) nanoparticles as a photocatalyst. Results show that the photocatalytic reaction occurs via the Langmuir-Hinshelwood mechanism and the photocatalysis kinetics in the confined microsystem (r = 0.359 min-1) is about 10 times larger than that in macrosystem (r = 0.033 min-1). In addition, the photocatalysis activity of the immobilized TiO2 nanoparticles in the microreactor exhibits good stability under flowing conditions. The present microchip device offers an interesting platform for screening of photocatalysts and exploration of photocatalysis mechanisms and kinetics.