Photobleaching Of Dye Research Articles

Use of Interfacial Interactions and Complexation of Carbon Dots to Construct Ultra-Robust and Efficient Photothermal Film From Micro-Carbonized Polysaccharides.

Solar energy conversion technologies, particularly solar-driven photothermal conversion, are both clean and manageable. Although much progress has been made in designing solar-driven photothermal materials, significant challenges remain, not least the photobleaching of organic dyes. To tackle these issues, micro-carbonized polysaccharide chains, with carbon dots (CDs) suspended from the chains, are conceived, just like grapes or tomatoes hanging from a vine. Carbonization of sodium carboxymethyl cellulose (CMC) produces just such a structure (termed CMC-g-CDs), which is used to produce an ultra-stable, robust, and efficient solar-thermal film by interfacial interactions within the CMC-g-CDs. The introduction of the CDs into the matrix of the photothermal material effectively avoided the problem of photobleaching. Manipulating the interfacial interactions (such as electrostatic interactions, van der Waals interactions, π-π stacking, and hydrogen bonding) between the CDs and the polymer chains markedly enhances the mechanical properties of the photothermal film. The CMC-g-CDs are complexed with Fe3+ to eliminate leakage of the photothermal reagent from the matrix and to solve the problem of poor water resistance. The resulting film (CMC-g-CDs-Fe) has excellent prospects for practical application as a photothermal film.

Enhanced long-term stability of stained microplastics with carbon nitride fluorescent polymer for tracking

Fluorescent labeling and imaging is a useful tool to track the temporal and spatial distribution of microplastics (MPs) with size and shape features. However, the limited fluorescently labeled MPs spheres, photo-bleaching and leaching of organic small molecular dyes hamper the tracking of MPs in the environment. In this work, phenyl-modified carbon nitride (PMCN) fluorescent polymer was prepared and applied for the staining of eight different types of MPs. The interface interaction mechanism between PMCN fluorescent polymer and MPs was investigated by the desorption experiments. The long-time photo-stability of the stained MPs against ultraviolet (UV) light irradiation, freeze-thaw cycles, various environmental factors and real environmental water samples was evaluated. The MPs stained with PMCN fluorescent polymer showed more robust stability than the common dye (Nile red) against the damage when exposure to UV light for 72 h, treatment with freeze-thaw cycles for 60 days, exposure to strong acid, strong alkali, high salinity, freshwater (river and lake) or seawater for 60 days. Results indicate PMCN fluorescent polymer is promising as a convenient, efficient and universal fluorescent tracer for visually tracking the migration and distribution of MPs in environmental water system.

Optical traps induce fluorophore photobleaching by two-photon excitation

Techniques combining optical tweezers with fluorescence microscopy have become increasingly popular. Unfortunately, the high-power, infrared lasers used to create optical traps can have a deleterious effect on dye stability. Previous studies have shown that dye photobleaching is enhanced by absorption of visible fluorescence excitation plus infrared trap photons, a process that can be significantly reduced by minimizing simultaneous exposure to both light sources. Here, we report another photobleaching pathway that results from direct excitation by the trapping laser alone. Our results show that this trap-induced fluorescence loss is a two-photon absorption process, as demonstrated by a quadratic dependence on the intensity of the trapping laser. We further show that, under conditions typical of many trap-based experiments, fluorescence emission of certain fluorophores near the trap focus can drop by 90% within 1 min. We investigate how photostability is affected by the choice of dye molecule, excitation and emission wavelength, and labeled molecule. Finally, we discuss the different photobleaching pathways in combined trap-fluorescence measurements, which guide the selection of optimal dyes and conditions for more robust experimental protocols.

Tetrameric Antibody Complexes and Affinity Tag Peptides for the Selective Immobilization and Imaging of Single Quantum Dots.

Colloidal semiconductor quantum dots (QDs) are of widespread interest as fluorescent labels for bioanalysis and imaging applications. Single-particle measurements have proven to be a very powerful tool for better understanding the fundamental properties and behaviors of QDs and their bioconjugates; however, a recurring challenge is the immobilization of QDs in a solution-like environment that minimizes interactions with a bulk surface. Immobilization strategies for QD-peptide conjugates are particularly underdeveloped within this context. Here, we present a novel strategy for the selective immobilization of single QD-peptide conjugates using a combination of tetrameric antibody complexes (TACs) and affinity tag peptides. A glass substrate is modified with an adsorbed layer of concanavalin A (ConA) that binds a subsequent layer of dextran that minimizes nonspecific binding. A TAC with anti-dextran and anti-affinity tag antibodies binds to the dextran-coated glass surface and to the affinity tag sequence of QD-peptide conjugates. The result is spontaneous and sequence-selective immobilization of single QDs without any chemical activation or cross-linking. Controlled immobilization of multiple colors of QDs is possible using multiple affinity tag sequences. Experiments confirmed that this approach positions the QD away from the bulk surface. The method supports real-time imaging of binding and dissociation, measurements of Förster resonance energy transfer (FRET), tracking of dye photobleaching, and detection of proteolytic activity. We anticipate that this immobilization strategy will be useful for studies of QD-associated photophysics, biomolecular interactions and processes, and digital assays.

Fluorescence Intermittency of Quantum Dot–Organic Dye Conjugates: Implications for Alternative Energy and Biological Imaging

Quantum dot (QD)-organic dye couple chromophores are topical due to their applications in biology, catalysis, and energy. The maximization of energy transfer efficiency can be guided by the underlying Förster or Dexter mechanisms; however, the impact of fluorescence intermittency must also be considered. Here we demonstrate that the average ⟨ton⟩ and ⟨toff⟩ times of dye acceptors in coupled QD-dye chromophores are substantially affected by the donors' blinking behavior. With regard to biological imaging, this effect beneficially minimizes the photobleaching of the acceptor dye. The implications for alternative energy are less encouraging as the acceptors' capacity to store energy, using ⟨ton⟩/⟨toff⟩ as a metric, was reduced by as much as ∼95%. These detrimental effects can be mitigated by suppressing QD blinking via surface treatment. This study also demonstrates several instances of the nonconformity of QD blinking dynamics to a power law distribution, as a robust examination of the off times reveals log-normal behavior that is consistent with the Albery model.

Optical gain and photo-bleaching of organic dyes, quantum dots, perovskite nanoplatelets and nanodiamonds

ABSTRACT Optical gain and resistance against photo-bleaching are key material parameters for practical applications in microlasers and microphotonics. Here, we present studies of optical amplification and photo-bleaching in a wide range of optical gain materials, including fluorescent dyes, quantum dots and rods, organic and inorganic room temperature polaritons, and nanodiamonds with emission range from 430 to 680 nm. We used amplified spontaneous emission (ASE) for each material to measure the optical gain. Robustness against photo-bleaching was determined by measuring the intensity of spontaneous emission of the material as a function of the number of excitation femtosecond pulses (106–1010) at various excitation energy densities. We show that pyrromethene laser dyes are the best organic emitters in terms of low excitation energy and good optical gain, whereas solid-state polariton materials are better in terms of high optical gain and stability. We found that all organic and inorganic optical gain materials bleach completely after 106–109 illumination pulses with typical 0.1–0.6 GW/cm2 peak power density, with an exception of nanodiamonds. We show that nanodiamonds are the only optical gain material that shows no photo-bleaching beyond the 1010 excitation pulses of 300 fs pulse duration and with 0.16 GW/cm2 peak power density.

Development of an optical thermography system using a pumped two-dye fluorescence technique

This work demonstrates the development of a novel backside thermography technique based on the temperature sensitivity of laser-induced fluorescence in flowing two-dye solutions. The approach utilizes visible light and optically transparent packaging materials to obtain spatially resolved transient thermal measurements. This makes it a relatively simple, inexpensive, and flexible approach for lab-scale experimental characterizations using economical cameras and optics. Additionally, both heating and cooling of the thermography stage can be achieved by passing temperature-controlled water through plastic packaging components. A custom-built experimental setup was designed, constructed, and used to study the performance of seven two-dye Rhodamine B (RhB)-Rhodamine 110 (Rh110) fluorescent solutions. The effect of dye concentration ratio on sensitivity, maximum frame rate, and excitation area was characterized. Using an optimal dye concentration of unity, it was shown that frame rates of 265–3200 Hz are achievable for excitation areas with diameters of 15.5–4.3 mm, respectively, using the current setup. The calibrated system was used to demonstrate in-situ measurements showing the importance of two-dye light compensation, as well as backside thermography using a simple droplet contact method to investigate temporal response. Experiments were conducted in the range of 25–55 °C with a spatial resolution of 30 µm. The experimental uncertainty of the temperature measurements was calculated to be ±2.1 °C and the technique's ability to account for error due to light fluctuations and non-uniform illumination was demonstrated. Finally, the effect of dye photobleaching during prolonged testing was studied and it was shown that photobleaching can be reduced, and even eliminated, by maintaining a nominal low flow rate of the pumped two-dye solution.

Hybrid structure design, preparation of Ag-GO SERS optical fiber probe and its chemical, electromagnetic enhancement mechanism

Incorporating Ag-graphene oxide hybrid material (Ag-GO) on optical fiber offers a novel approach for flexible ultrasensitive detection in biomedical and environmental areas. In this work, Ag-GO surface-enhanced Raman scattering (SERS) optical fiber probe was synthesized by a SnCl2-sensitized solvothermal method and modified Hummer’s method. After optimizing SERS test parameters, SERS performance of the fiber probes synthesized by different synthetic procedures of GO, Ag-GO hybrid structures and GO concentration are compared. The obtained GO shows superior ability to quench fluorescence by reducing the photobleaching of dyes. GO with high oxidation degree is preferred. Compared to Ag and hybrid structures including GO/Ag and GO/Ag/GO, Ag/GO fiber probe exhibits strongest enhancement due to π-π stacking and charge transfer process from lone pair of electrons on GO to the molecules. Ag/GO fiber probe with 5 mg/mL GO displays strongest SERS enhancement. Apart from chemical enhancement, it is worthwhile to note that GO is also found to selectively enhance specific vibrations regarding π bond or lone pair of electrons. The fiber probe shows high sensitivity (10−9 M R6G), good reproducibility (RSD of 3.5%) and good stability. Moreover, the enhanced area of Ag-GO fiber probe occurs in core area and cladding edge. The enhancement mechanism of Ag-GO fiber probe is that, as GO provides an additional route for excited dyes to decay through its Fermi level, photobleaching of dyes is reduced by charge transfer process. The enhancement is a synergic effect of chemical enhancement of GO, electromagnetic enhancement of silver and waveguide propagation of optical fiber.

Precise Detection and Visualization of Cyclooxygenase-2 for Golgi Imaging by a Light-Up Aggregation-Induced Emission-Based Probe

Precise Detection and Visualization of Cyclooxygenase-2 for Golgi Imaging by a Light-Up Aggregation-Induced Emission-Based Probe

Cerium oxide nano-particles application for self-cleaning building material

Photocatalytic degradation has gained attraction in the self-cleaning construction materials applications because of its ability to be used to protect surface of cementitious materials and minimize the negative effects of environmental conditions on building, and to remove the dyes and other organic pollutants from water and wastewater. In this study, the influence of nano-Ceria particles on the photocatalytic degradation of methylene blue dye of cement mortar samples was investigated. In this work, nanoceria was prepared via simple co-precipitation method. Nanoceria is a powerful photocatalyst due to its high visible-light absorptivity, strong redox ability, high oxygen storage/release capacity, eco-friendly nature, photostability and cost effectiveness. Nano ceria showed a photocatalytic and self-cleaning activity in the presence of sun radiation. Nano ceria particles were characterized by X-ray diffraction, High resolution transmission electron microscope, and Ultra-violet visible spectroscopy. The presence of active oxygen species detected on nano- ceria surface was identified as cause of the photobleaching of dyes in presence of UV-irradiated.

Heterogeneously Strained Tissue Collagen Resists Collagenase Degradation where Strains are High

Tissue homeostasis reflects a balance of synthesis and degradation, and past studies of homogeneously stressed tissues and collagen materials indicate collagen degradation is suppressed by strain. Such processes are studied here in isolated tendons and in beating embryonic hearts. In tendon fascicles, collagen fibrils primarily orient along the tension axis but heterogeneous strains and gradients were induced using two different deformation modes i.e. heterogeneous (using three-point bending plus adhesion) and uniaxial, with patterned photo-bleaching of a fluorescent dye and collagen-binding peptide on fascicles used to measure strain.

Bayesian Classification and Modeling of Single Molecule Fluorescence Colocalization Images

Multi-wavelength single-molecule fluorescence colocalization methods allow elucidation of complex biochemical reaction mechanisms. However, analysis of colocalization data is an intrinsically challenging and time-consuming problem for multiple reasons. First, to minimize dye photobleaching, images frequently are collected at low signal-to-noise ratios, making it challenging to discriminate real fluorescent spots from noise. Second, transient non-specific interactions of the binder molecule with the surface of the microscope slide can give rise to both false-positive and false-negative detection.

More Symmetrical "Hot Spots" Ensure Stronger Plasmon-Enhanced Fluorescence: From Au Nanorods to Nanostars.

Plasmon-enhanced fluorescence (PEF) is considered to be a powerful signal amplification technology to overcome intrinsic shortcomings of photobleaching and brightness of the traditional fluorescent dyes. Nevertheless, exploitation of PEF-based probes for bioimaging application is still at a very early stage. In this work, a simple but powerful gold nanostar (Au NST)@SiO2-based PEF probe with 20 symmetric "hot spots" was developed for highly sensitive "lighting up" in situ imaging of intracellular microRNAs (miRNAs). By regulating the thickness of the silica shell, the distance between Au NSTs and fluorescent dyes was controlled, and the optimum fluorescence enhancement (21-fold) was obtained with the silica shell thickness of approximately 22 nm. Thanks to the 20 more powerful "hot spots" that can produce stronger localized electric fields, the Au NST-based PEF probe exhibits stronger PEF effects than the traditional plasmonic nanostructures such as gold nanorods (Au NRs), gold nanobipyramids (Au NBPs), and triangular gold nanoprisms (Au NPRs), resulting in high sensitivity and improved detection limit (LOD) of 0.21 pM for miRNA-21 analysis. Moreover, not only cancer cells (MCF-7 and Hela) and normal cells (L02) with distinct miRNA-21 expression levels can be discriminated but also tumor cells in co-cultured mixtures can be recognized, indicating its promising potential in clinical diagnosis.

Dual-dye systems comprising activatable fluorescein dye and hydrophobic or hydrophilic Cy5 reference fluorophore for ratiometric drug delivery monitoring

Ratiometric fluorescence detection in biological samples in vitro and in vivo mitigates several issues in conventional single-signal measurements such as light absorption and scattering, dye photobleaching, and instrumental parameters. Herein, two new systems for ratiometric monitoring of drug delivery are synthesized and investigated. These systems comprise a non-fluorescent fluorescein-based dye, Flu, bound to the anticancer drug, azatoxin (Aza), through a hydrolytically cleavable acrylate ester linker and either hydrophobic (Cy5h) or hydrophilic (Cy5s) pentamethine cyanine dye providing a red-light reference signal. Upon drug release, the green Flu fluorescence notably increases enabling ratiometric tracking of drug release events. The spectral properties of the developed conjugates and fluorescently monitored drug release rates are investigated in biologically relevant media with respect of the Cy5 dye hydrophilicity.

Fluorescent labeling of plasmid DNA for gene delivery: Implications of dye hydrophobicity on labeling efficiencies and nanoparticle size

Covalent fluorescent labels are important tools for monitoring the in vitro and in vivo localization of plasmid DNA nanoparticles, but must meet several criteria including high DNA labeling efficiencies and minimal impact on nanoparticle size. We developed a novel fluorescent labeling strategy utilizing an aryl azide photolabel conjugated to a short cationic peptide to label plasmid DNA with Cyanine 5 and sulfo-Cyanine 5. Using a simple camera flash apparatus, photolabel-peptide-dyes can be conjugated to DNA in minutes with preservation of DNA structure and minimal dye photobleaching. The addition of two anionic sulfonates to the Cyanine 5 core greatly improved labeling efficiencies from ~13 to ~53% and mitigated PEGylated polyacridine peptide-DNA nanoparticle size increases over a range of labeling densities. Comparison of our sulfo-Cyanine 5 peptide label to the Mirus Bio Label IT-Cy5 kit revealed that while both did not affect nanoparticle sizes appreciably, labeling efficiencies with our conjugate were higher, possibly due to the higher positive charge density on the peptide linker. The results from this work provide important considerations for choosing fluorophore tags to track DNA nanoparticles.

Low-Noise Fluorescent Infrared Detection of Single-Cell Cardiac Action Potentials

Optical recordings with voltage-sensitive fluorescent dyes enable non-invasive action potential (AP) measurements, which are useful for high-throughput drug screening. However, optical measurements are hampered by dye rundown, photobleaching, and phototoxicity. Long-lasting infrared dyes overcome these limitations, but optical recordings from single-cells are difficult due to low signal-to-noise ratios (SNR). Using the voltage-sensitive infrared dye, Di-4-ANBDQBS (Cytovolt1), we developed an optical system consisting of matched hardware, biological, and dye components to produce a low-noise fluorescent infrared detection system for single-cell AP recording. We combined a high-sensitivity photodiode-based detector, a high-stability LED excitation source, and an optimized optical filter set for enhanced single-cell optical AP recordings. We validated our system by correlating membrane voltage to fluorescence under single-cell patch clamp, and showed the fluorescence signal is highly-linear with membrane voltage. We also optimized dye concentration and staining method along with emission/excitation wavelengths to yield maximum ΔF/F and thus maximum SNR. To correlate membrane voltages to optical voltage signals, we performed simultaneous patch clamp and optical recordings on HEK293 cells and human induced pluripotent stem cell derived cardiomyocytes (hIPSC-CMs). Low-noise recordings, with minimal dye rundown, were obtained from single-cells. Optical signals were recorded in response to a voltage ramp, and averaged R2 values from linear fits of fluorescence versus voltage were 0.88+0.04, 0.97+0.007, 0.97+0.004 and 0.98+0.004 for 50, 75, 100 and 150 μM of Di‑4‑ANBDQBS, respectively, in HEK293 cells. ΔF/F measurements were 10.5+0.3%, 14.9+1.8%, 13.8+2.5% and 13.6+3.5% for 50, 75, 100 and 150 μM of dye, respectively. For hiPSC-CMs, ΔF/F measurements from simultaneous voltage and fluorescence of APs were up to 23.1% per 100 mV. Taken together, these data demonstrate the feasibility of high fidelity single cell voltage measurements in a simple system not requiring photomultiplier tubes or laser excitation.

Optical Imaging of Isolated Murine Ventricular Myocytes.

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

Parallel Acceleration on Removal of Optical Mapping Baseline Wandering.

Optical mapping measurements on hearts stained with fluorescent dyes is imagining method widely accepted and recognized as a tool to study complex spatial-temporal dynamics of cardiac electro-physiology. One shortcoming of the method is baseline wandering in obtained fluorescence signals as signals relevant to transmembrane potential (Vm) change and free intracellular calcium concentration ([Ca]i +2), the two most used dyes, are calculated as a relative signal change in respect to the fluorescence baseline. These changes are small fractional changes often smaller than 10 %. Baseline fluorescence drifts due to dye photo-bleaching, heart contraction/movement artifacts, and stability of the excitation light source over time. Depending on experimental instrumentation, recording duration, signal to noise levels and study aims of the optical imagining, many research groups adopted their own techniques tailored to a specific experimental data. Here we present a technique based on finite impulse response (FIR) filters with paralleled acceleration implemented on GPUs and multi-core CPU, in MATLAB.

Quantitative determination of cellular [Na+] by fluorescence lifetime imaging with CoroNaGreen.

Fluorescence lifetime imaging microscopy (FLIM) with fluorescent ion sensors enables the measurement of ion concentrations based on the detection of photon emission events after brief excitation with a pulsed laser source. In contrast to intensity-based imaging, it is independent of dye concentration, photobleaching, or focus drift and has thus been successfully employed for quantitative analysis of, e.g., calcium levels in different cell types and cellular microdomains. Here, we tested the suitability of CoroNaGreen for FLIM-based determination of sodium concentration ([Na+]) inside cells. In vitro measurements confirmed that fluorescence lifetimes of CoroNaGreen (CoroNaFL) increased with increasing [Na+]. Moreover, CoroNaFL was largely independent of changes in potassium concentration or viscosity. Changes in pH slightly affected FL in the acidic range (pH ≤ 5.5). For intracellular determination of [Na+], HEK293T cells were loaded with the membrane-permeable form of CoroNaGreen. Fluorescence decay curves of CoroNaGreen, derived from time-correlated single-photon counting, were approximated by a bi-exponential decay. In situ calibrations revealed a sigmoidal dependence of CoroNaFL on [Na+] between 0 and 150 mM, exhibiting an apparent K d of ∼80 mM. Based on these calibrations, a [Na+] of 17.6 mM was determined in the cytosol. Cellular nuclei showed a significantly lower [Na+] of 13.0 mM, whereas [Na+] in perinuclear regions was significantly higher (26.5 mM). Metabolic inhibition or blocking the Na+/K+-ATPase by removal of extracellular K+ caused significant [Na+] increases in all cellular subcompartments. Using an alternative approach for data analysis ("Ratio FLIM") increased the temporal resolution and revealed a sequential response to K+ removal, with cytosolic [Na+] increasing first, followed by the nucleus and finally the perinuclear regions. Taken together, our results show that CoroNaGreen is suitable for dynamic, FLIM-based determination of intracellular [Na+]. This approach thus represents a valuable tool for quantitative determination of [Na+] and changes thereof in different subcellular compartments.

Dynamic Measurement of Nanoflows: Realization of an Optofluidic Flow Meter to the Nanoliter-per-Minute Scale.

The ultimate performance of flow-based measurements in microfluidic systems is currently limited by their accuracy at the nanoliter-per-minute scale. Improving such measurements (especially in contexts that require continuous monitoring) is challenging because of constraints associated with shrinking system geometries and limitations imposed by making precise measurements of smaller quantities in real time. A particularly interesting limit is the relative uncertainty as flow approaches zero, which diverges for most measurement methods. To address these problems, we have developed an optofluidic measurement system that can deliver and record light in a precise interrogation region of a microfluidic channel. The system utilizes photobleaching of fluorophore dyes in the bulk flow and can identify zero flow to better than 1 nL/min absolute accuracy. The technique also provides an independent method for determining nonzero flow rates based on a robust scaling relationship between the fluorescence emission and flow. Together, these two independent approaches enable precise measurement of flow to within 5% accuracy down to 10 nL/min and validation of flow control to within 5% uncertainty down to 2 nL/min. We also demonstrate that our technique can be used to extend a calibrated flow meter well below its specified range (e.g., 500 nL/min) and to make dynamic measurements of similar relative uncertainties to the calibrated meter, which would have otherwise expanded significantly in this regime.