Methods and Applications in Fluorescence最新文献

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.

This work describes the relationship between the complex of photosystem I and photosystem II in the senescence process of rice leaves observed through changes in the optical response. We studied three varieties of rice plants at different aging times using time-resolved photoluminescence to measure the time decay of the emission, and stationary photoluminescence, to measure the emission wavelength. The spectra obtained with the former technique were fitted with decreasing exponential functions. Two relaxation times were obtained, one ranging between 1.0 and 1.7 ns, and the other, from 5.0 to 10.5 ns. They are associated with the electron's deexcitation of PSI and PSII, respectively, and these decay times increase as the leaf senescence process takes place. The spectra obtained with stationary photoluminescence were fitted with Voigt functions. These spectra exhibit two main peaks around 683 and 730 nm, which could be associated mainly with PSII and PSI emissions, respectively. The PSI de-excitation exhibits higher dispersive processes because chlorophyll-a molecules in it move away from each other, decreasing their concentration. Therefore, it takes longer for electrons to recombine during photosynthesis, as seen in the time-resolve response. Articulating the results of both photoluminescence techniques, the changes in the response of the photosystems of the living rice leaves during senescence are evidenced.

Polyvinylpyrrolidone (PVP) can be used to produce upconversion nanoparticles (UCNPs) in an advantageous manner, i.e. at modest temperatures in open-to-air conditions with simple hotplate and flask apparatus. However, the influence of PVP parameters on the formation of UCNPs has not been previously investigated. In this exploratory study, we establish that PVP molecular weight and relative amount of PVP can greatly influence the morphology and diameter of NaYF₄:Yb,Er UCNPs produced via the PVP-assisted route. At nominal amounts of PVP, varying the molecular weight of PVP in synthesis between 10,000 g mol^-1(PVP10), 40,000 g mol^-1(PVP40), and 55,000 g mol^-1(PVP55), had minimal effect on UCNP morphology, whereas reducing the quantity of PVP10 and PVP40 in the reaction to 10% of the nominal amount resulted in two notable effects: (1) the generation of a greater range of UCNP diameters and (2) the production of an unexpected sub-population of rhombus-shaped UCNPs. Bulk and individual nanoparticle analysis indicates that all UCNP morphologies were cubic (α-phase) crystal structure and consisted of NaYF₄:Yb,Er. Optical emission properties exhibited only modest green and red luminescence emission ratio when PVP parameters were varied. However, separately produced PVP40 NaYF₄:Yb,Tm UCNPs exhibited a much more intense and dual-band blue/red emission. This exploratory work demonstrates that tailoring PVP content in synthesis of UCNPs can greatly alter morphology of UCNPs produced and should be carefully considered in experimental design. However, the underlying mechanisms of action of the role PVP plays in this synthesis remain unclear. Ultimately, significant further work is still required to fully elucidate the relevant chemistry to achieve full control of PVP-UCNP synthesis.

Two different pairs of laser dyes, Rhodamine-110 (Rh-110)/Rhodamine-6G (Rh-6G) and Rhodamine-19 (Rh-19)/Rhodamine-B (Rh-B) (the first dye in each pair as a donor and the second as an acceptor) were impregnated in silica samples prepared by the sol-gel method and spectroscopically studied using absorption and steady-state fluorescence techniques. The critical transfer distance (R₀), actual distance (r) between the donor and acceptor, overlap integral [J(υ¯)], FRET (fluorescence resonance energy transfer) efficiency (E), and antenna effect efficiency (AE) were investigated in detail based on the variation in acceptor concentration. The FRET efficiency, antenna effect efficiency, and actual donor-acceptor distance for Rh-110/Rh-6G and Rh-19/Rh-B dye pairs corresponding to acceptor concentration ranges (3.83 to 7.65) × 10^-5M l^-1and (3.71 to 8.34) × 10^-5M l^-1, respectively, were found to be in the ranges of 57.38% to 74.89%, 36.97% to 24.13%, 5.44 nm to 4.77 nm, and 77.01%. Furthermore, maximum FRET efficiencies of 85.68% and 87.63% and antenna effect efficiencies of 36.97% and 40.95% for Rh-110/Rh-6G and Rh-19/Rh-B, respectively, were also reported. Our results demonstrate the superior FRET efficiency of Rh-19/Rh-B over Rh-110/Rh-6G dye pair in sol-gel glasses, while the antenna effect efficiency of Rh-110/Rh-6G is higher than that of Rh-19/Rh-B for the same donor to acceptor (D/A) ratio. Finally, Rh-110/Rh-6G is a better energy harvester than the Rh-19/Rh-B dye pair at the common D/A ratio. These results are explained in terms of molecular structure similarity, polarity, and rigidity of donor and acceptor.

The wavelength-ratiometric techniques gain increasing popularity in fluorescence probing and sensing for providing inner reference to output signal and removing instrumental artefacts, in this way increasing the sensitivity and reliability of assays. Recent developments demonstrate that such approach can allow achieving much more, with the application of broad range of novel molecular and nanoscale fluorophores (luminophores), exploring the whole power of photophysical and photochemical effects and using extended range of assay formats. Simplicity of detection and potentially rich content of output data allows realizing these techniques in different simplified, miniaturized and multiplexing devices. The latter issues are discussed in Pt. II of these series.

The problem of enhanced molecular emission in close proximity to dielectric and metallic interfaces is of great importance for many physical and biological applications. Here we present an exact treatment of the problem from the view point of classical electromagnetism. Self-consistent analytical theory of the surface enhanced fluorescence (SEF) is developed for configurations consisting of an emitter in proximity to core-shell metal-dielectric nanoparticles. The dependence of the fluorescence enhancement on the excitation laser and fluorescence frequencies and distance of the emitter to the nanoparticle interface are studied. The developed theory predicts enhanced fluorescence at intermediate distances as well as emission quenching into non-radiative surface plasmon (SP) modes dominating the response for short distances. The conditions for optimal emission enhancement for two core-shell configurations are determined and a comparison to published experimental data is performed showing a good correspondence between theory and experiment. The developed model can be applied toward analyzes and optimizations of various applications related to SP enhance fluorescence spectroscopy.

The wavelength-ratiometric techniques demonstrate strong advantages in fluorescence sensing and imaging over techniques employing variations of intensity at single wavelength. We present different possibilities for realization of these advantages in different simplified, miniaturized and multiplexing devices. They include the smartphone-based detection systems and strips, in which the color changes are observed with naked eye. The array-based techniques and different immunoassays withλ-ratiometric detection demonstrate strongly increased stability and sensitivity. The application areas extend from on-site monitoring of environment and point-of-care diagnostics to testing in personal need. Selected examples of sensing different analytes in chemical and biological systems demonstrate multiple possibilities of coupling the analyte-sensor interaction with the generation ofλ-ratiometric output signal. Among them, simultaneous detection of several analytes and performing logical operations that can be useful in analysis. Finally, the benefits of multicolor ratiometric fluorescence imaging are demonstrated by visualization the functionally important parameters of biological membranes.