A luminescence lifetime assisted ratiometric fluorimeter for biological applications.
Ontology highlight
ABSTRACT:
SUBMITTER: Lam H
PROVIDER: S-EPMC2803713 | biostudies-other | 2009 Dec
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Rapid, multiplexed, sensitive and specific identification and quantitative detection of nitric oxide (NO) are in great demand in biomedical science. Herein, a novel multifunctional probe based on the intramolecular LRET (luminescence resonance energy transfer) strategy, TRP-NO, was designed for the highly sensitive and selective ratiometric and luminescence lifetime detection of lysosomal NO. Before reaction with NO, the emission of the rhodamine moiety in TRP-NO is switched off, which prevents the LRET process, so that the probe emits only the long-lived Tb3+ luminescence. However, upon reaction with NO, accompanied by the turn-on of rhodamine emission, the LRET from the Tb3+-complex moiety to rhodamine moiety occurs, which results in a remarkable increase of the rhodamine emission and decrease of the Tb3+ emission. After the reaction, the intensity ratio of the rhodamine emission to the Tb3+ emission, I565/I540, was found to be 28.8-fold increased, and the dose-dependent enhancement of the I565/I540 value showed a good linearity upon the increase of NO concentration. In addition, a dose-dependent luminescence lifetime decrease was distinctly observed between the average luminescence lifetime of the probe and NO concentration, which provides a ∼10-fold contrast window for the detection of NO. These unique properties allowed TRP-NO to be conveniently used as a time-gated luminescence probe for the quantitative detection of NO using both luminescence intensity ratio and luminescence lifetime as signals. The applicability of TRP-NO for ratiometric time-gated luminescence imaging of NO in living cells was investigated. Meanwhile, dye co-localization studies confirmed a quite precise distribution of TRP-NO in lysosomes by confocal microscopy imaging. Furthermore, the practical applicability of TRP-NO was demonstrated by the visualization of NO in Daphnia magna. All of the results demonstrated that TRP-NO could serve as a useful tool for exploiting and elucidating the function of NO at sub-cellular levels with high specificity, accuracy and contrast.
| S-EPMC5384565 | biostudies-literature
Project description:In luminescence thermometry enabling temperature reading at a distance, an important challenge is to propose new solutions that open measuring and material possibilities. Responding to these needs, in the nanocrystalline phosphors of yttrium oxide Y2O3 and lutetium oxide Lu2O3, temperature-dependent emission of trivalent terbium Tb3+ dopant ions was recorded at the excitation wavelength 266 nm. The signal of intensity decreasing with temperature was monitored in the range corresponding to the 5D4 → 7F6 emission band. On the other hand, defect emission intensity obtained upon 543 nm excitation increases significantly at elevated temperatures. The opposite thermal monotonicity of these two signals in the same spectral range enabled development of the single band ratiometric luminescent thermometer of as high a relative sensitivity as 4.92%/°C and 2%/°C for Y2O3:Tb3+ and Lu2O3:Tb3+ nanocrystals, respectively. This study presents the first report on luminescent thermometry using defect emission in inorganic phosphors.
| S-EPMC7407274 | biostudies-literature
Project description:Luminescence thermometry in biomedical sciences is a highly desirable, but also highly challenging and demanding technology. Numerous artifacts have been found during steady-state spectroscopy temperature quantification, such as ratiometric spectroscopy. Oppositely, the luminescence lifetime is considered as the most reliable indicator of temperature thermometry because this luminescent feature is not susceptible to sample properties or luminescence reabsorption by the nanothermometers themselves. Unfortunately, this type of thermometer is much less studied and known. Here, the thermometric properties of Yb3+ ions in Nd0.5RE0.4Yb0.1PO4 luminescent temperature probes were evaluated, aiming to design and optimize luminescence lifetime based nanothermometers. Temperature dependence of the luminescence lifetimes is induced by thermally activated phonon assisted energy transfer from the 2F5/2 state of Yb3+ ions to the 4F3/2 state of Nd3+ ions, which in turn is responsible for the significant quenching of the Yb3+:2F5/2 lifetime. It was also found that the thermal quenching and thus the relative sensitivity of the luminescent thermometer can be intentionally altered by the RE ions used (RE = Y, Lu, La, and Gd). The highest relative sensitivity was found to be S R = 1.22% K-1 at 355 K for Nd0.5Y0.4Yb0.1PO4 and it remains above 1% K-1 up to 500 K. The high sensitivity and reliable thermometric performance of Nd0.5La0.4Yb0.1PO4 were confirmed by the high reproducibility of the temperature readout and the temperature uncertainty being as low as δT = 0.05 K at 383 K.
| S-EPMC9418974 | biostudies-literature
Project description:Time-resolved flow cytometry represents an alternative to commonly applied spectral or intensity multiplexing in bioanalytics. At present, the vast majority of the reports on this topic focuses on phase-domain techniques and specific applications. In this report, we present a flow cytometry platform with time-resolved detection based on a compact setup and straightforward time-domain measurements utilizing lifetime-encoded beads with lifetimes in the nanosecond range. We provide general assessment of time-domain flow cytometry and discuss the concept of this platform to address achievable resolution limits, data analysis, and requirements on suitable encoding dyes. Experimental data are complemented by numerical calculations on photon count numbers and impact of noise and measurement time on the obtained lifetime values.
| S-EPMC6233182 | biostudies-literature
Project description:The NIR excitation and large anti-Stokes shift of upconversion nanoparticles (UCNPs) have made them an ideal choice as biological nanoprobes. A key challenge in the field is to confer biorecognition units to UCNPs so that they can be used to probe specific targets in biological systems. While various agents have been combined with UCNPs to meet such a challenge, most studies are limited to small molecules, while biomolecules such as metalloproteins that possess much higher affinity and selectivity for metal ions have not been explored. Herein we demonstrate that fusion of zwitterion-coated UCNPs with azurin, a member of a family of redox-active copper proteins called cupredoxins that play important roles in diverse biological functions, can serve as an ideal platform for the label-free upconversion luminescence sensing of Cu2+ with a ratiometric response. The selectively binding of apo-azurin with Cu2+ induce a significant absorbance at about 625 nm, and hence decreases the red emission of the UCNPs. In contrast, the green emission of the UCNPs remains constant and acts as an internal standard reference for the ratiometric sensing of Cu2+. This approach opens a new window for the development of assays for biosensing based on a combination of specific metalloproteins with UCNPs.
| S-EPMC7081933 | biostudies-literature
Project description:The luminescence dynamics in ensembles of nanocrystals are complicated by a variety of processes, including the size-dependence of the radiative and non-radiative rates in inhomogeneous broadened samples and interparticle interactions. This results in a non-exponential decay, which for the specific case of silicon nanocrystals (SiNCs) has been widely modeled with a Kohlrausch or "stretched exponential" (SE) function. We first derive the population decay function for a luminescence decay following exp[-?(t/?)?]. We then compare the distributions and mean times calculated by assuming that either the luminescence decay or the population decay follows this function and show that the results are significantly different for ? much below 1. We then apply these two types of SE functions as well as other models to the luminescence decay data from two thermally grown SiNC samples with different mean sizes. The mean lifetimes are strongly dependent on the experimental setup and the chosen fitting model, none of which appears to adequately describe the ensemble decay dynamics. Frequency-resolved spectroscopy (FRS) techniques are then applied to SiNCs in order to extract the lifetime distribution directly. The rate distribution has a half width of ~?0.5 decades and mainly resembles a somewhat high-frequency-skewed lognormal function. The combination of TRS and FRS methods appear best suited to uncovering the luminescence dynamics of NC materials having a broad emission spectrum.
| S-EPMC6261909 | biostudies-other
Project description:We report the development of a genetically encodable and ratiometic pH probe named "pHlash" that utilizes Bioluminescence Resonance Energy Transfer (BRET) rather than fluorescence excitation. The pHlash sensor-composed of a donor luciferase that is genetically fused to a Venus fluorophore-exhibits pH dependence of its spectral emission in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification in vivo. Its spectral ratio response is H(+) specific; neither Ca(++), Mg(++), Na(+), nor K(+) changes the spectral form of its luminescence emission. Moreover, it can be used to image pH in single cells. This is the first BRET-based sensor of H(+) ions, and it should allow the approximation of pH in cytosolic and organellar compartments in applications where current pH probes are inadequate.
| S-EPMC3419163 | biostudies-literature
Project description:We report a ratiometric temperature imaging method based on Mn luminescence from Mn-doped CdS-ZnS nanocrystals (NCs) with controlled doping location, which is designed to exhibit strong temperature dependence of the spectral lineshape while being insensitive to the surrounding chemical environment. Ratiometric thermometry on the Mn luminescence spectrum was performed by using Mn-doped CdS-ZnS core-shell NCs that have a large local lattice strain on the Mn site, which results in the enhanced temperature dependence of the bandwidth and peak position. The Mn luminescence spectral lineshape is highly robust with respect to the change in the polarity, phase and pH of the surrounding medium and aggregation of the NCs, showing great potential in temperature imaging under chemically heterogeneous environment. The temperature sensitivity (?IR/IR = 0.5%/K at 293 K, IR = intensity ratio at two different wavelengths) is highly linear in a wide range of temperatures from cryogenic to above-ambient temperatures. We demonstrate the surface temperature imaging of a cryo-cooling device showing a temperature variation of >200 K by imaging the luminescence of the NC film formed by simple spin coating, taking advantage of the environment-insensitive luminescence.
| S-EPMC3661212 | biostudies-literature
Project description:In contrast to traditional short-lived fluorescent probes, long-lived phosphorescent probes based on transition-metal complexes can effectively eliminate unwanted background interference by using time-resolved luminescence imaging techniques, such as photoluminescence lifetime imaging microscopy. Hence, phosphorescent probes have become one of the most attractive candidates for investigating biological events in living systems. However, most of them are based on single emission intensity changes, which might be affected by a variety of intracellular environmental factors. Ratiometric measurement allows simultaneous recording of two separated wavelengths instead of measuring mere intensity changes and thus offers built-in correction for environmental effects. Herein, for the first time, a soft salt based phosphorescent probe has been developed for ratiometric and lifetime imaging of intracellular pH variations in real time. Specifically, a pH sensitive cationic complex (C1) and a pH insensitive anionic complex (A1) are directly connected through electrostatic interaction to form a soft salt based probe (S1), which exhibits a ratiometric phosphorescent response to pH with two well-resolved emission peaks separated by about 150 nm (from 475 to 625 nm). This novel probe was then successfully applied for ratiometric and lifetime imaging of intracellular pH variations. Moreover, quantitative measurements of intracellular pH fluctuations caused by oxidative stress have been performed for S1 based on the pH-dependent calibration curve.
| S-EPMC6006953 | biostudies-literature
Project description:To monitor the lateral segregation of lipids into liquid-ordered (Lo) and -disordered (Ld) phases in lipid membranes, environment-sensitive dyes that partition in both phases but stain them differently have been developed. Of particular interest is the dual-color F2N12S probe, which can discriminate the two phases through the ratio of its two emission bands. These bands are associated with the normal (N(∗)) and tautomer (T(∗)) excited-state species that result from an excited-state intramolecular proton transfer. In this work, we investigated the potency of the time-resolved fluorescence parameters of F2N12S to discriminate lipid phases in model and cell membranes. Both the long and mean lifetime values of the T(∗) form of F2N12S were found to differ by twofold between Ld and Lo phases as a result of the restriction in the relative motions of the two aromatic moieties of F2N12S imposed by the highly packed Lo phase. This differed from the changes in the ratio of the two emission bands between the two phases, which mainly resulted from the decreased hydration of the N(∗) form in the Lo phase. Importantly, the strong difference in lifetimes between the two phases was preserved when cholesterol was added to the Ld phase. The two phases could be imaged with high contrast by fluorescence lifetime imaging microscopy (FLIM) on giant unilamellar vesicles. FLIM images of F2N12S-labeled live HeLa cells confirmed that the plasma membrane was mainly in the Lo-like phase. Furthermore, the two phases were found to be homogeneously distributed all over the plasma membrane, indicating that they are highly mixed at the spatiotemporal resolution of the FLIM setup. Finally, FLIM could also be used to sensitively monitor the change in lipid phase upon cholesterol depletion and apoptosis.
| S-EPMC4457243 | biostudies-literature