Methods and Applications in Fluorescence最新文献

We studied the effect of annealing on the luminescence of Coumarin 106 (C106) in poly (vinyl alcohol) films (PVA films). The samples and reference polymer films were treated at temperatures between 100 °C and 150 °C (212 F and 302 F) for various times. After cooling and smoothing, the samples and references were measured at room temperature. We observed that the PVA polymer (reference films) changes its optical properties with annealing at higher temperatures, affecting the baselines in absorption and the backgrounds in emission measurements. This requires precise background subtractions and control of the signal-to-noise ratio. Whereas the fluorescence intensity of C106 in PVA films modestly decreases with annealing, the phosphorescence depends dramatically and progressively increases by many folds. The fluorescence quantum yields and lifetimes decrease with the annealing, which suggests an increase in the non-radiative processes in the singlet excited state S₁. The increase in the phosphorescence intensities results from increased intersystem crossing (ISC), which also decreases fluorescence. We also studied the effect of annealing on phosphorescence with the directly excited triplet state of C106. In this case, two processes are affected by annealing, S₀→T₁absorption and T₁→S₀phosphorescence. The long-wavelength excitation (475 nm) avoids PVA polymer excitation. The phosphorescence lifetime decreases with annealing while the phosphorescence intensity increases. These changes suggest that the radiative rate of T₁→ S₀increases with annealing.

Efficient and rapid detection of angiotensin-converting enzyme (ACE) activity is important for preventing hypertension and the discovery of new angiotensin-converting enzyme inhibitors (ACEI). In this work, a single-excitation and double-emission biomass-derived carbon quantum dots (CQDs) was prepared and applied for ratiometric fluorescence detection of ACE. Fresh banyan leaves were extracted with ethanol and acetone, and the extracted solution was used as the precursor to produce the carbon quantum dots (BL-CQDs) with single-excitation and double-emission properties. The synthesized BL-CQDs is about 1.7 nm, has a graphene-like structure, contains a variety of hydrophilic functional groups on the surface, and has good fluorescence properties. Its fluorescence intensity ratio (I₆₇₇/I₄₆₀) is linear with ACE activity in the range of 0.02-0.8 U l^-1. The regression equation is△F=2.5371C_ACE-0.0311. The method was successfully applied to the determination of ACE activity in pig lung and human serum, and the inhibitory efficiency of the flavonoid extract and captopril tablets on ACE activity was also investigated, which can be applied to the screening of ACEI. The survival rate and fluorescence imaging of Bel-7404 cells under the condition of high concentration BL-CQDs showed BL-CQDs had low cytotoxicity and good biocompatibility. These results indicate that the BL-CQDs can be used as an excellent fluorescent probe, providing a new method for screening ACE activity and plant-derived ACEI.

PIFE was first used as an acronym for protein-induced fluorescence enhancement, which refers to the increase in fluorescence observed upon the interaction of a fluorophore, such as a cyanine, with a protein. This fluorescence enhancement is due to changes in the rate ofcis/transphotoisomerisation. It is clear now that this mechanism is generally applicable to interactions with any biomolecule. In this review, we propose that PIFE is thereby renamed according to its fundamental working principle as photoisomerisation-related fluorescence enhancement, keeping the PIFE acronym intact. We discuss the photochemistry of cyanine fluorophores, the mechanism of PIFE, its advantages and limitations, and recent approaches to turning PIFE into a quantitative assay. We provide an overview of its current applications to different biomolecules and discuss potential future uses, including the study of protein-protein interactions, protein-ligand interactions and conformational changes in biomolecules.

Fluorescence microscopy can provide valuable information about cell interior dynamics. Particularly, mean squared displacement (MSD) analysis is widely used to characterize proteins and sub-cellular structures' mobility providing the laws of molecular diffusion. The MSD curve is traditionally extracted from individual trajectories recorded by single-particle tracking-based techniques. More recently, image correlation methods like iMSD have been shown capable of providing averaged dynamic information directly from images, without the need for isolation and localization of individual particles. iMSD is a powerful technique that has been successfully applied to many different biological problems, over a wide spatial and temporal scales. The aim of this work is to review and compare these two well-established methodologies and their performance in different situations, to give an insight on how to make the most out of their unique characteristics. We show the analysis of the same datasets by the two methods. Regardless of the experimental differences in the input data for MSD or iMSD analysis, our results show that the two approaches can address equivalent questions for free diffusing systems. We focused on studying a range of diffusion coefficients between D = 0.001μm²s^-1and D = 0.1μm²s^-1, where we verified that the equivalence is maintained even for the case of isolated particles. This opens new opportunities for studying intracellular dynamics using equipment commonly available in any biophysical laboratory.

The challenge of building a highly reliable contactless temperature probe with high sensitivity, good temperature-induced color discriminability, and economical synthesis has prompted the research community to work in the field of rare-earth-based luminescence thermometry. Moreover, the fast-growing market for optoelectronic devices has increased the demand for tunable color-emitting phosphors. In this study, Dy³⁺/Eu³⁺co-doped SrMoO₄phosphors were developed as tunable color-emitting source and dual-mode luminescence thermometer. A facile and cost-effective auto-combustion method was used to synthesize the phosphors. Our work demonstrates a viable scheme for tailoring the emission of single-phase phosphors by precisely controlling the dopant concentrations and by modulating excitation wavelength. The overall emission is tuned from greenish-yellow to white and greenish-yellow to reddish-orange. A detailed energy transfer process from the host to the Ln³⁺ions and between the Ln³⁺ions is discussed. Further, anti-thermal quenching in the emission of Dy³⁺ion is observed when excited with 297 nm. The dual-mode luminescence thermometry has been studied by analyzing the fluorescence intensity ratio of Dy³⁺and Eu³⁺ions upon excitation at 297 nm. The maximum relative sensitivity value for 4% Eu³⁺co-doped SrMoO₄:4%Dy³⁺phosphor is 1.46% K^-1at 300 K. Furthermore, the configurational coordinate diagram is presented to elucidate the nature of temperature-dependent emission. Therefore, our research opens up new avenues for the development of color-tunable luminescent materials for various optoelectronic and temperature-sensing applications.

Melamine has been intentionally added into food products to increase the protein count at less cost, especially in dairy products for infant resulting in serious adverse effects on health of consumers. Therefore, this study aimed to develop a method to quantify melamine in dairy products based on the change of fluorescent properties of carbon dots (CDs) as sensing probe. CDs with green-fluorescent emission were synthesized from citric acid and urea under microwave irradiation. The synthesized CDs emitted fluorescence at the maximum wavelength of 538 nm with excitation wavelength of 410 nm. Thus, they provided high sensitivity and selectivity on melamine detection by which fluorescent emission of the CDs was increasingly quenched upon increasing melamine concentrations. Optimal conditions for melamine determination using the CDs was under pH 6, volume ratio between CDs and sample of 2:8 and reaction time of 15 min. The developed method provided high precision of melamine determination with less than 5% of %RSD (n = 5), wide detection range from 1.0 to 200.0 ppm, and high sensitivity with limit of detection (LOD) of 0.47 ppm and limit of quantification (LOQ) of 1.56 ppm, which is within the regulated level by the Food and Drug Administration of the United States for melamine in dairy products. Several analytical characterization techniques were conducted to elucidate the reaction mechanism between CDs and melamine, and the hydrogen bonding interaction was proposed.

Photo-induced dark transient states of fluorophores can pose a problem in fluorescence spectroscopy. However, their typically long lifetimes also make them highly environment sensitive, suggesting fluorophores with prominent dark-state formation yields to be used as microenvironmental sensors in bio-molecular spectroscopy and imaging. In this work, we analyzed the singlet-triplet transitions of fluorescein and three synthesized carboxy-fluorescein derivatives, with one, two or four bromines linked to the anthracence backbone. Using transient state (TRAST) spectroscopy, we found a prominent internal heavy atom (IHA) enhancement of the intersystem crossing (ISC) rates upon bromination, inferred by density functional theory calculations to take place via a higher triplet state, followed by relaxation to the lowest triplet state. A corresponding external heavy atom (EHA) enhancement was found upon adding potassium iodide (KI). Notably, increased KI concentrations still resulted in lowered triplet state buildup in the brominated fluorophores, due to relatively lower enhancements in ISC, than in the triplet decay. Together with an antioxidative effect on the fluorophores, adding KI thus generated a fluorescence enhancement of the brominated fluorophores. By TRAST measurements, analyzing the average fluorescence intensity of fluorescent molecules subject to a systematically varied excitation modulation, dark state transitions within very high triplet yield (>90%) fluorophores can be directly analyzed under biologically relevant conditions. These measurements, not possible by other techniques such as fluorescence correlation spectroscopy, opens for bio-sensing applications based on high triplet yield fluorophores, and for characterization of high triplet yield photodynamic therapy agents, and how they are influenced by IHA and EHA effects.

In this study, Robinia hispida L leaves (RH) was used as a precursor for the first time to synthesize fluorescent carbon dots (CDs) with stable blue fluorescence by a single-step hydrothermal synthesis method. Notably, the innovative approach eliminates the necessity for toxic chemicals or hazardous substances, marking a significant advancement in the field. The synthesized CDs demonstrate CDs demonstrates the predominance of spherical shapes with an average size of 11.63 ± 1.92 nm. The CDs not only exhibit an enhanced fluorescent efficiency with a relatively high quantum yield of up to 6.8%, but they also possess the potential for direct utilization in the selective determination of Hg(II) through fluorescence quenching, even without any functionalization. Under the optimized conditions at a pH of 7.0, a robust linear correlation was found to exist between the fluorescence intensity and the concentration of Hg (II) within the range of 5-17.5μM, exhibiting a detection limit (3σ) of 1.5μM. Additionally, this methodology was effectively employed to successfully detect Hg (II) ions in various aqueous samples, including tap water, spring water, drinking water, and a certified reference material (CRM-SA-C Sandy Soil C). The spike recoveries of 97.6%-101.6% with less than 2.7% variability were performed on all samples.

The cell membrane has a fundamental role in the cell life cycle but there's still much to be learned about its heterogeneous structure, regulation, and protein interaction. Additionally, the protein-membrane interaction is often overlooked when studying specific protein dynamics. In this work, we present a new tool for a better understanding of protein dynamics and membrane function using live cells and fast non-invasive techniques without the need for individual particle tracking. To this end, we used the 2D-pair correlation function (2D-pCF) to study protein interactions across cellular membranes. We performed numerical simulations and confocal experiments using a GAP-mEGFP fusion construct known to interact with the plasmatic membrane. Our results demonstrate that based on a quantitative correlation analysis as the 2D pair correlation of the signal intensities, is possible to characterize protein-membrane interactions in live systems and real-time. Combining experimental and numerical results this work presents a new powerful approach to the study of the dynamic protein-membrane interaction.

The survival rate of oral squamous cell carcinoma (OSCC) patients is very poor, but it can be improved using highly sensitive, specific, and accurate techniques. Autofluorescence and fluorescence techniques are very sensitive and helpful in cancer screening; being directly linked with the molecular levels of human tissue, they can be used as a quantitative tool for cancer detection. Here, we report the development of multi-modal autofluorescence and fluorescence imaging and spectroscopic (MAF-IS) smartphone-based systems for fast and real-time oral cancer screening. MAF-IS system is indigenously developed and offers the advantages of being a low-cost, handy, non-contact, non-invasive, and easily operable device that can be employed in hospitals, including low-resource settings. In this study, we report the results of 43 individuals with 28 OSCC and 15 oral potentially malignant disorders (OPMDs), i.e., epithelial dysplasia and oral submucous fibrosis, using the developed devices. We observed a red shift in fluorescence emission spectrain vivo. We found red-shift of 7.72 ± 6 nm, 3 ± 4.36 nm, and 1.33 ± 0.47 nm in the case of OSCC, epithelial dysplasia, and oral submucous fibrosis, respectively, compared to normal. The results were compared with histopathology and found to be consistent. Further, the MAF-IS system provides results in real-time with higher accuracy and sensitivity compared to devices using a single modality. Our system can achieve an accuracy of 97% with sensitivity and specificity of 100% and 94.7%, respectively, even with a smaller number of patients (28 patients of OSCC). The proposed MAF-IS device has great potential for fast screening and diagnosis of oral cancer in the future.