Methods and Applications in Fluorescence最新文献

The stoichiometry of molecular components within supramolecular biological complexes is often an important property to understand their biological functioning, particularly within their native environment. While there are well established methods to determine stoichiometryin vitro, it is presently challenging to precisely quantify this propertyin vivo,especially with single molecule resolution that is needed for the characterization stoichiometry heterogeneity. Previous work has shown that optical microscopy can provide some information to this end, but it can be challenging to obtain highly precise measurements at higher densities of fluorophores. Here we provide a simple approach using already established procedures in single-molecule localization microscopy (SMLM) to enable precise quantification of stoichiometry within individual complexes regardless of the density of fluorophores. We show that by focusing on the number of fluorophore detections accumulated during the quasi equilibrium-state of this process, this method yields a 50-fold improvement in precision over values obtained from images with higher densities of active fluorophores. Further, we show that our method yields more correct estimates of stoichiometry with nuclear pore complexes and is easily adaptable to quantify the DNA content with nanodomains of chromatin within individual chromosomes inside cells. Thus, we envision that this straightforward method may become a common approach by which SMLM can be routinely employed for the accurate quantification of subunit stoichiometry within individual complexes within cells.

Green, one-pot, quick, and easily synthesized nitrogen and sulfur co-doped carbon quantum dots (N,S-CDs) were obtained from cheap and readily available chemicals (sucrose, urea, and thiourea) using a microwave-assisted approach in about 4 min and utilized as a turn-off fluorescent sensor for estimation of natamycin (NAT). First, the effect of N and S doping on the microwave-synthesized CDs' quantum yield was carefully studied. CDs derived from sucrose alone failed to produce a high quantum yield; then, to increase the quantum yield, doping with heteroatoms was carried out using either urea or thiourea. A slight increase in quantum yield was observed upon using thiourea with sucrose, while an obvious enhancement of quantum yield was obtained when urea was used instead of thiourea. Surprisingly, using a combination of urea and thiourea together results in N,S-CDs with the highest quantum yield (53.5%), uniform and small particle size distribution, and extended stability. The fluorescent signal of N,S-CDs was quenched upon addition of NAT due to inner filter effect and static quenching in a manner that allowed for quantitative determination of NAT over a range of 0.5-10.0μg ml^-1(LOD = 0.10μg ml^-1). The N,S-CDs were applicable for determination of NAT in aqueous humor, eye drops, different environmental water samples, and bread with excellent performance. The selectivity study indicated excellent selectivity of the prepared N,S-CDs toward NAT with little interference from possibly interfering substances. In-silico toxicological evaluation of NAT was conducted to estimate its long-term toxicity and drug-drug interactions. Finally, the preparation of N,S-CDs, and analytical procedure compliance with the green chemistry principles were confirmed by two greenness assessment tools.

Antibodies have gained considerable importance in laboratory and clinical settings. Currently, antibodies are extensively employed for the diagnosis and treatment of several human diseases. Herein, using targeted and cell immunisation approaches, we developed and characterised an antibody clone, DWH24. We found that DWH24 is an IgMκtype antibody that enables excellent visualisation and quantification of dead cells using immunofluorescence, fluorescence microscopy, and flow cytometry. This property was proved by the spontaneous cell death of several tumour cell lines and stimulated T cells, as well as after chemo- and photodynamic therapy. Unlike conventional apoptosis and cell death markers, DWH24 binding occurred in a Ca²⁺- and protein-independent manner and enabled live imaging of cell death progress, as shown using time-lapse microscopy. The binding specificity of DWH24 was analysed using a human proteome microarray, which revealed a complex response profile with very high spot intensities against various proteins, such as tropomyosin variants and FAM131C. Accordingly, DWH24 can be employed as a suitable tool for the cost-effective and universal analysis of cell death using fluorescence imaging and flow cytometry.

A facile, simple, green and sensitive spectrofluorometric method was developed for determination of the calcimimetic drug cinacalcet hydrochloride. It is used for the treatment of hyperparathyroidism. The drug showed high native fluorescence intensity at 320 nm after excitation at 280 nm. The method was linear over the range of 5.0-400.0 ng ml^-1with excellent correlation (R²= 0.9999). Limit of detection (LOD) and limit of quantitation (LOQ) values were 1.19 and 3.62 ng ml^-1, respectively. The percentage recovery was found to be 100.42% ± 1.39 (n=8). The proposed method was successfully applied for determination of cinacalcet in spiked human plasma samples with % recoveries of (87.23 to 109.69%). Two recent greenness metrics (GAPI and Analytical Eco-Scale) were chosen to prove the eco-friendly nature of the method. Furthermore, the proposed method was successfully applied to dissolution study of commercial cinacalcet tablets. The interference likely to be introduced by some commonly co-administrated drugs such as metoprolol and itraconazole was studied; the tolerance limits were calculated.

The small molecular drugs pharmacodynamics and pharmacokinetics could be affected by human serum albumin (HSA) transport, so we studied the interaction between HSA and the widely used anti-ischemic agent, trimetazidine (TMZ), using different approaches. As shown by synchronous fluorescence spectroscopy, the interaction affects the microenvironment confirmation around tyrosine residues. The site-competitive experiments showed that TMZ had an affinity toward subdomain III A (site II) of HSA. The enthalpy and entropy changes (ΔH and ΔS), which were 37.75 and 0.197 K J mol^-1, respectively, showed that the predominant intermolecular interactions are hydrophobic forces. According to FTIR research, the interaction between HSA and TMZ caused polypeptide carbonyl-hydrogen bonds to rearrange. The HSA esterase enzyme activity was decreased with TMZ. Docking analysis confirmed the site-competitive experiments and thermodynamic results. This study demonstrated that TMZ interacted with HSA, and the structure and function of HSA were influenced by TMZ. This study could aid in understanding the pharmacokinetics of TMZ and provide basic data for safe use.

Urine is a highly complex fluorescent system, the fluorescence of which can be affected by many factors, including the often-ignored initial urine concentration in comprehensive fluorescent urine analysis. In this study, a total urine fluorescent metabolome profile (uTFMP) was created as a three-dimensional fluorescence profile of serial synchronous spectra of urine diluted by geometric progression. uTFMP was generated using software designed for this purpose after recalculating the 3D data concerning the initial urine concentration. It can be presented as a contour map (top view) or as a more illustrative and straightforward simple curve, thus usable in various medicinal applications.

The present work investigated the influence of different halides on the excited state dynamics of 6-methoxyflavone (6MF) in an aqueous solution with steady-state and time-resolved techniques. On successive addition of I^-and Br^-ions, the fluorescence of 6MF quenched significantly, whereas the respective ions do not change the maximum fluorescence band. Fluorescence of 6MF was quenched 66% by I^-ions and 34% by Br^-ions. In a pure aqueous medium, both the H-bonded: CT and protonated species of 6MF participate in the quenching of fluorescence. The quenching process was categorized by Stern-Volmer (S-V) and Lehrer equations. Quenching parameters such as K_SV, K_SV-Land k_qwere higher for I^-ions than Br^-ions. The decrease in fluorescence intensity and a reduction in fluorescence lifetime suggested the dynamic nature of quenching by I^-ions following the electron transfer mechanism. Fluorescence quenching of 6MF has also been observed in the acidic medium in the presence of different halides. Thus, the study reveals that 6MF is responsive towards I^-ions in a wide range of pH, specifically in a purely aqueous environment (pH∼7), hence important for sensing/detection applications.

Multi-color fluorescence imaging is a powerful tool for studying the spatial relationships and interactions among sub-cellular structures in biological specimens. However, if improperly corrected, geometrical distortions caused by mechanical drift, refractive index mismatch, or chromatic aberration can lead to lower image resolution. In this paper, we present an extension of the image processing framework of Scipion by integrating a protocol called OFM Corrector, which corrects geometrical distortions in real-time using a B-spline-based elastic continuous registration technique. Our proposal provides a simple strategy to overcome chromatic aberration by digitally re-aligning color channels in multi-color fluorescence microscopy images, even in 3D or time. Our method relies on a geometrical calibration, which we do with fluorescent beads excited by different wavelengths of light and subsequently registered to get the elastic warp as a reference to correct chromatic shift. Our software is freely available with a user-friendly GUI and can be broadly used for various biological imaging problems. The paper presents a valuable tool for researchers working in light microscopy facilities.

Enhanced green fluorescence protein (EGFP) is a fluorescent tag commonly used in cellular and biomedical applications. Surprisingly, some interesting photochemical properties of EGFP have remained unexplored. Here we report on two-photon-induced photoconversion of EGFP, which can be permanently converted by intense IR irradiation to a form with a short fluorescence lifetime and spectrally conserved emission. Photoconverted EGFP thus can be distinguished from the unconverted tag by the time-resolved detection. Nonlinear dependence of the two-photon photoconversion efficiency on the light intensity allows for an accurate 3D localization of the photoconverted volume within cellular structures, which is especially useful for kinetic FLIM applications. For illustration, we used the two photon photoconversion of EGFP for measurements of redistribution kinetics of nucleophosmin and histone H2B in nuclei of live cells. Measurements revealed high mobility of fluorescently tagged histone H2B in the nucleoplasm and their redistribution between spatially separated nucleoli.