Methods and Applications in Fluorescence最新文献

Cell viability assessment plays a crucial role in biological research, pharmaceutical development, and toxicological identification. Here, we used GelRed, a sensitive and safer nucleic acid dye, to selectively label dead cells with red fluorescence (FL) thus distinguishing dead cells from live ones. Further more, the combined use of GelRed and SYTO 9 (another nucleic acid dye) enabled the clear differentiation in FL spectra between the two physiological statuses. The GelRed and SYTO 9 concentrations were optimized to obtain the highest FL ratio of dead to live cells. The GelRed/SYTO 9-based double staining could quantify the cell viability through flow cytometry analysis, with a good correlation between the detected and theoretical dead cell ratios. Compared with traditional prodium iodide (PI) staining, the GelRed/SYTO 9-based double staining showed high accuracy in quantifying dead cell of low levels. The as-developed staining method could be used in biomedical research to accurately measure the cytotoxic effect of various substances in living cells. .

Molybdenum disulfide quantum dots (MoS2 QDs) is a new type of graphite like nanomaterial, which exhibited well chemical stability, unique fluorescence characteristics, and excellent biocompatibility. The conventional hydrothermal synthesis of MoS2 generally requires a long-term reaction at high temperature and high pressure. Herein, we have developed a simple and fast MoS2 QDs synthesis scheme using microwave heating, and further modified the surface of MoS2 QDs using 3-aminophenylboronic acid. The 3- aminophenylboronic acid modified MoS2 QDs (B-MoS2 QDs) were further coated by a zinc-based metal-organic backbone (ZIF-8) in a solution containing zinc ions and 2-methylimidazolium. The constructed nanohybrid B-MoS2@ZIF-8 were successfully applied to the visualization and rapid detection of bilirubin based on the ratiometric fluorescence changes. The linear range for bilirubin detection is 0.2-75 μmol•L-1, and detection limit is 0.017 μmol•L-1.

The current culture-based bacterial detection technique is time-consuming and requires an extended sample preparation methodology. We propose the potential of surface-enhanced Raman spectroscopy (SERS) and surface plasmon-enhanced auto-fluorescence spectroscopy (SPEAS) for the label-free identification and quantification of bacterial pathogens at low concentrations collecting its unique auto-fluorescence and Raman signatures utilising highly anisotropic three-dimensional nanostructures of silver nano dendrites (Ag-NDs). The SERS data facilitates qualitative bacterial identification using the spectral features coming from the bacterial cell wall compound, and the SPEAS data was utilised to gain unique auto-fluorescence spectra present on the bacterial cell wall with enhanced quantification. The enhancement of Raman and auto-fluorescence signals of Ag-NDs were first evaluated using rhodamine 6g (R6G) as a probe molecule that exhibits a significant enhancement of 106 and limit of detection (LOD) of 10-12 M for SERS and 15 fold intensity enhancement and LOD of 10-15 M for SPEAS measurements. Further, the SERS and SPEAS measurements of bacterial pathogens, such as Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), using the Ag-NDs were recorded, and the results exhibit high auto-fluorescence and Raman signal intensity for both the samples up to 100 cfu/ml for both modalities. The significant photon count and distinct emission range in SPEAS measurements of bacteria enables accurate quantification. Therefore, the comprehensive investigation of plasmonic enhancement of Ag-NDs for SPEAS and SERS techniques provides complementary information about molecules to enable accurate and quick identification and quantification of pathogens.

We studied absorption and fluorescence as well as room temperature phosphorescence (RTP) of 4-methylumbelliferone (4MU) in poly (vinyl alcohol) (PVA) films. We focused our study on the long-wavelength basic form of 4MU with absorption centered at 375 nm. The strong fluorescence with a quantum yield of above 70% appears at ∼430 nm. The fluorescence anisotropy of 4MU-doped PVA film is high, reaching a value of about 0.3. The emission with gated detection shows a broad phosphorescence spectrum with a peak at ∼510 nm and a residual delayed fluorescence at 430 nm. The excitation spectra for fluorescence and phosphorescence roughly follows 4MU absorption. The phosphorescence lifetime of 4MU is remarkably long, almost 3 s. 4MU excitation and emission phosphorescence anisotropies are low, very close to zero.

The escalating prevalence of hospital-acquired infections poses a critical challenge for healthcare systems worldwide. Effective management requires rapid identification of pathogens and their antibiotic resistance profiles. In this study, we utilized the photoconvertible mEos4b protein, which transitions from green to red fluorescence upon blue light exposure, to distinguish live from dead bacteria. The mEos4b gene was cloned into a prokaryotic vector and expressed inEscherichia coliBL21. The Minimum Inhibitory Concentration (MIC) of the transgenic bacteria was determined for five antibiotics, followed by a post-antibiotic effect assessment over a two-hour exposure period. The optimal photoconversion time for mEos4b was established as 90 s, and confocal microscopy was used to visualize live (green) and dead (red) cells post-exposure. The mEos4b-TR system proved highly specific, accurately distinguishing live and dead bacteria without producing false positives, even in control groups, which is a common issue in commercial live-dead kits. By relying on cellular metabolic activity rather than dyes, this system minimizes nonspecific interactions and contamination, making it more reliable than traditional methods prone to false readings. These results highlight the potential of the mEos4b-TR system as a superior alternative for rapid, precise bacterial viability assessments, particularly in determining antibiotic susceptibility. This preliminary study demonstrates the system's differentiation of viable and non-viable cells, suggesting its potential application in future studies involving novel antibacterial agents to refine antibiotic sensitivity testing.

Linker-Evolved-Group-Optimized-Lipophosphonoxins (LEGO-LPPO) are small synthetic modular peptidomimetics with promising antimicrobial activity. The LEGO-LPPO mechanism of antibacterial action has been determined to be the depolarization and disruption of bacterial membranes. Their modular nature is advantageous for fine tuning their biological properties. In order to optimize the structure of LEGO-LPPO even further, it is important to understand the interaction of LEGO-LPPO with bacterial membranes at the molecular level. In this work, we present the synthesis of five LEGO-LPPO (designated as1_naph2-4-G to5_naph2-4-G) molecules bearing fluorescent naphtylethyl moieties and their usage in the study of LEGO-LPPO behaviour in the membrane. Our goal was to characterize fluorescently labelled LEGO-LPPO under conditions that do not completely disrupt the membrane, mostly in the form of membrane-bound monomers. We observed the intramolecular interactions of hydrophobic modules of1_naph2-4-G in the buffer by detecting dynamic naphthyl excimers and their disappearance after1_naph2-4-G bind into the membranes. In the membrane, the molecule1_naph2-4-G slightly affects the membrane fluidity of DOPG membranes above the phase transition. The naphthyl fluorophore itself has fast and almost unrestricted rotation around ethylene linking groups (r_inf= 0.010), which indicates a considerable chaotropic effect of the hydrophobic modules of1_naph2-4-G at the given depth of the membrane.1_naph2-4-G proved to be a useful model for observing the interaction of LEGO-LPPO antibiotics with the phospholipid bilayer enabling us to decipher its effects on membrane state and dynamics; its binding and penetration into the membrane, its structure and the particular depth that it occupies.

Detection of autofluorescence parameters is a useful approach to gain insight into the physiological state of plants and algae, but the effect of reabsorption hinders unambiguous interpretation ofin vivodata. The exceptional morphological features ofNitellopsis obtusamade it possible to measure autofluorescence spectra along single internodal cells and estimate relative changes in autofluorescence intensity in selected spectral regions at room temperatures, avoiding the problems associated with thick or optically dense samples. The response of algal cells to controlled white light and DCMU herbicide was analyzed by monitoring changes in peak FL intensity at 680 nm and in F680/F750 ratio. Determining the association between the selected spectral FL parameters revealed an exponential relationship, which provides a quantitative description of photoinduced changes. The ability to discern the effect of DCMU not only in the autofluorescence spectra of dark-adapted cells, but also in the case of light-adapted cells, and even after certain doses of excess light, suggests that the proposed autofluorescence analysis ofN. obtusamay be useful for detecting external stressors in the field.

Cesium lead halide perovskite (CsPbX₃; X = Cl, Br, I) nanocrystals showing intense band-edge emission and high photoluminescence quantum yield are known to be a potential candidate for application in optoelectronic devices. However, controlling toxicity due to the presence of Pb²⁺in lead-based halide perovskites is a major challenge for the environment that needs to be tackled cautiously. In this work, we have partially replaced Pb²⁺with Mn²⁺ions in the CsPb(Cl/Br)₃nanocrystals and investigated their impact on the structural and optical properties. The Rietveld refinement shows that CsPbCl₂Br nanocrystals possess a cubic crystal structure withPm3̅mspace group, the Mn²⁺doping results in the contraction of the unit cell. The CsPb(Cl/Br)₃: Mn nanocrystals show a substantial change in the optical properties with an additional emission band at ∼588 nm through a d-d transition, changing the emission color from blue to pink. Here, a didodecyldimethylammonium bromide (DDAB) ligand that triggers both anion and ligand exchange in the CsPb(Cl/Br)₃: Mn nanocrystals have been used to regulate the exchange reaction and tune the emission color of halide perovskites by changing the peak position and the PL intensities of band-edge and Mn²⁺defect states. We have also shown that oleic acid helps in the desorption of oleylamine capping from the CsPb(Cl/Br)₃: Mn nanocrystal surfaces and DDAB, resulting in the substitution of Cl^-with Br^-as well as provides capping with shorter branched length ligand which led to increase in the overall PL intensity by many folds.

Wines are complex mixtures of chemical compounds with broad and overlapping absorption and emission spectral features in the UV and visible spectral regions, making them challenging to study with conventional optical spectroscopic techniques. Multidimensional fluorescence spectroscopies correlate fluorescence spectra with other degrees of freedom, and have proven useful for studying complex molecular systems, offering a pathway for the analysis of wines utilising their inherent fluorescence. Here we employ steady-state excitation-emission matrix (EEM) and time-resolved fluorescence spectral measurements to investigate representative commercial white and red wine samples and a fluorescent 'model' wine base. Combining these multidimensional measurement methods provides information on the emission characteristics of the components that wines contain. This investigation illustrates the potential for multidimensional fluorescence techniques as diagnostic tools for the wine industry.