Methods and Applications in Fluorescence最新文献

Coherently coupled pseudoisocyanine (PIC) dye aggregates have demonstrated the ability to delocalize electronic excitations and ultimately migrate excitons with much higher efficiency than similar designs where excitations are isolated to individual chromophores. Here, we report initial evidence of a new type of PIC aggregate, formed through heterogeneous nucleation on DNA oligonucleotides, displaying photophysical properties that differ significantly from previously reported aggregates. This new aggregate, which we call the super aggregate (SA) due to the need for elevated dye excess to form it, is clearly differentiated from previously reported aggregates by spectroscopic and biophysical characterization. In emission spectra, the SA exhibits peak narrowing and, in some cases, significant quantum yield variation, indicative of stronger coupling in cyanine dyes. The SA was further characterized with circular dichroism and atomic force microscopy observing unique features depending on the DNA substrate. Then by integrating an AlexaFluor^TM647 (AF) dye as an energy transfer acceptor into the system, we observed mixed energy transfer characteristics using the different DNA. For example, SA formed with a rigid DNA double crossover tile (DX-tile) substrate resulted in AF emission sensitization. While SA formed with more flexible non-DX-tile DNA (i.e. duplex and single strand DNA) resulted in AF emission quenching. These combined characterizations strongly imply that DNA-based PIC aggregate properties can be controlled through simple modifications to the DNA substrate's sequence and geometry. Ultimately, we aim to inform rational design principles for future device prototyping. For example, one key conclusion of the study is that the high absorbance cross-section and efficient energy transfer observed with rigid substrates made for better photonic antennae, compared to flexible DNA substrates.

The resolution achievable with the established super-resolution fluorescence nanoscopy methods, such as STORM or STED, is in general not sufficient to resolve protein complexes or even individual proteins. Recently, minimal photon flux (MINFLUX) nanoscopy has been introduced that combines the strengths of STED and STORM nanoscopy and can achieve a localization precision of less than 5 nm. We established a generally applicable workflow for MINFLUX imaging and applied it for the first time to a bacterial molecular machinein situ, i.e., the injectisome of the enteropathogenY. enterocolitica. We demonstrate with a pore protein of the injectisome that MINFLUX can achieve a resolution down to the single molecule levelin situ. By imaging a sorting platform protein using 3D-MINFLUX, insights into the precise localization and distribution of an injectisome component in a bacterial cell could be accomplished. MINFLUX nanoscopy has the potential to revolutionize super-resolution imaging of dynamic molecular processes in bacteria and eukaryotes.

We have studied the evolution of keratin intrinsic fluorescence as an indicator of its glycation. Steady-state and time-resolved fluorescence of free keratin and keratin-glucose samples were detected in PBS solutionsin vitro. The changes in the fluorescence response demonstrate that the effect of glucose is manifest in the accelerated formation of fluorescent cross-links with an emission peak at 460 nm and formation of new cross-links with emission peaks at 525 nm and 575 nm. The fluorescence kinetics of these structures is studied and their potential application for the detection of long-term complications of diabetes discussed.

A simpleα-cyanostilbene-functioned salicylaldehyde-based Schiff-base probe, which exhibited outstanding 'aggregation-induced emission and excited state intramolecular proton transfer (AIE + ESIPT)' emission in solution, aggregation and solid states, was synthesized in high yield of 87%. Its solid-states with different morphologies emitted different fluorescence after crystallization in EtOH/H₂O (1/2, v/v) mixtures or pure EtOH solvent. Besides, it exhibited an obvious spectro-photometrical fluorescence quenching for highly selective sensing of Co²⁺in THF/water system (ƒ_w= 60%, pH = 7.4), accompanied by an intense green fluorescence turn-off behavior under UV_365nmillumination. The binding stochiometry between the ligand and Co²⁺was found to be 2:1, and the detection limit (DL) was calculated to be 0.41 × 10^-8M. In addition, it could be applied to detect Co²⁺in real water samples and on silica gel testing strip.

A novel ionic conjugated polymer fluorescent probe (IP) containing fluorenyls and imines was synthesized through a simple condensation method for the rapid detection of Cu²⁺. The probe exhibited a selective quenching response toward Cu²⁺over other common metal ions in phosphate-buffered saline (PBS) at a pH of 7. Moreover, the fluorescence intensity ofIPwas linearly proportional to the concentration of Cu²⁺when it ranged from 1 to 10μM, and the limit of detection for Cu²⁺was 0.45μM. Furthermore, the quenched fluorescence ofIPby Cu²⁺was recovered after adding amino acids or ethylenediaminetetraacetic acid (EDTA), indicating that the probe could be recycled and is promising to reduce the detection cost of Cu²⁺in real samples.

The Er³⁺/Yb³⁺doped La₂O₃phosphor samples were synthesized by the combustion method and then photoluminescence and photoacoustic spectroscopic studies were done. Prepared samples were annealed at 800 °C, 1000 °C and 1300 °C and all samples were found in pure hexagonal phase as confirmed by XRD analysis. From FE-SEM images it is found that particle size increases with increase in annealing temperature. The frequency upconversion emission spectra of samples were recorded by exciting the sample with 980 nm diode laser and maximum emission intensity is obtained for the sample annealed at 1000 °C for 2 h. A photoacoustic cell was designed and wavelength dependent photoacoustic spectra were measured. The effect of sample storage time on radiative and non-radiative emission properties of sample was checked by measuring upconversion emission and photoacoustic spectra, simultaneously. It is observed that the emission intensity and photoacoustic signal both decreases with time. The maximum photoacoustic signal is obtained around 974 nm wavelength and it indicates its potential for photo-thermal therapy using infrared excitation.

Hyperspectral imaging (HSI) is a paramount technique in biomedical science, however, unmixing and quantification of each spectral component is a challenging task. Traditional unmixing relies on algorithms that need spectroscopic parameters from the fluorescent species in the sample. The phasor-based multi-harmonic unmixing method requires only the empirical measurement of the pure species to compute the pixel-wise photon fraction of every spectral component. Using simulations, we demonstrate the feasibility of the approach for up to 5 components and explore the use of adding a 6th unknown component representing autofluorescence. The simulations show that the method can be successfully used in typical confocal imaging experiments (with pixel photon counts between 10¹and 10³). As a proof of concept, we tested the method in living cells, using 5 common commercial dyes for organelle labeling and we easily and accurately separate them. Finally, we challenged the method by introducing a solvatochromic probe, 6-Dodecanoyl-N,N-dimethyl-2-naphthylamine (LAURDAN), intended to measure membrane dynamics on specific subcellular membrane-bound organelles by taking advantage of the linear combination between the organelle probes and LAURDAN. We succeeded in monitoring the membrane order in the Golgi apparatus, Mitochondria, and plasma membrane in the samein-vivocell and quantitatively comparing them. The phasor-based multi-harmonic unmixing method can help expand the outreach of HSI and democratize its use by the community for it does not require specialized knowledge.

Six luminescent, bright red Eu(III) complexes with aβ-keto-carboxylic acid as prime ligand and N-donor aromatic systems as auxillary ligand were synthesised via ecologically efficient grinding method. The distinctive red peak (⁵D₀ → ⁷F₂) of Eu(III) ion is exhibited in emission spectra of all complexes. The luminescent properties of complexes were analysed through decay time, color coordinates, luminescence efficiency and Judd Ofelt parameters. The value of Ω₂is found to be higher than Ω₄which indicated hypersensitive nature of⁵D₀ → ⁷F₂transition. The results established the complexes as a strong contender for red light emitting display devices. The fluorescence branching ratios, stimulated emission cross section, gain band width and optical gain showed the good lasing strength of⁵D₀ → ⁷F₂transition of complexes. The complexes exhibited decent thermal stability and have optical energy band gap value in semiconductor range, thus can have relevance in optoelectronic devices. Energy transfer mechanism was investigated for complexes which affirmed the efficacious transfer of energy from ligands to Eu(III) ion. The synthesised complexes were also assayed for antimicrobial and antioxidant properties. All complexes are reported to show better antioxidant behaviour than the prime ligand and also exhibited upstanding antibacterial activities.