Search

Single-particle tracking (SPT) is a powerful technique to elucidate the behavior of biomolecules inside or on the surface of living cells in real time. SPT is based on labeling the component of interest with a phosphor or a light-scattering gold nanoparticle, and visualizing its motion by real time microscopy. However, SPT generally suffers from suboptimal performance of phosphors. For example, fluorescent dyes have poor photostability, while quantum dots suffer from sporadic blinking that hampers track acquisition and interpretation. Light-scattering gold nanoparticles require custom experimental setups and relatively large particle size for tracking purposes. In recent years, upconverting nanoparticles (UCNPs) have emerged as a promising phosphor that enables backgroundless anti-Stokes luminescence imaging. They are also attractive for SPT applications due to their extreme photostability and non-blinking luminescence. Previously, SPT has been used to monitor UCNP endocytosis in cells. However, targeted SPT with UCNPs that specifically bind to biomolecules of interest has not yet been achieved. In this work, we provided a first proof-of-concept experiment of targeted SPT with UCNPs and showed the superior performance of UCNPs compared to organic dyes and quantum dots. As a benchmark system to assess the performance of UCNPs, we tracked FcεRI receptors on the surface of RBL-2H3 cell membranes using UCNP-IgE conjugates and obtained good agreement with data from the literature. Importantly, the versatility of the biotin-streptavidin architecture of our system allows UCNPs to be used to track a wide range of targets. Overall, this report lays the foundation for UCNP applications in targeted SPT.

Ensemble and single particle studies of the excitation power density (P)-dependent upconversion luminescence (UCL) of core and core-shell β-NaYF4:Yb,Er upconversion nanoparticles (UCNPs) doped with 20% Yb3+ and 1% or 3% Er3+ performed over a P regime of 6 orders of magnitude reveal an increasing contribution of the emission from high energy Er3+ levels at P > 1 kW/cm2. This changes the overall emission color from initially green over yellow to white. While initially the green and with increasing P the red emission dominate in ensemble measurements at P < 1 kW/cm2, the increasing population of higher Er3+ energy levels by multiphotonic processes at higher P in single particle studies results in a multitude of emission bands in the ultraviolet/visible/near infrared (UV/vis/NIR) accompanied by a decreased contribution of the red luminescence. Based upon a thorough analysis of the P-dependence of UCL, the emission bands activated at high P were grouped and assigned to 2–3, 3–4, and 4 photonic processes involving energy transfer (ET), excited-state absorption (ESA), cross-relaxation (CR), back energy transfer (BET), and non-radiative relaxation processes (nRP). This underlines the P-tunability of UCNP brightness and color and highlights the potential of P-dependent measurements for mechanistic studies required to manifest the population pathways of the different Er3+ levels.

A systematic study of the luminescence properties of monodisperse β-NaYF4: 20% Yb3+, 2% Er3+ upconversion nanoparticles (UCNPs) with sizes ranging from 12–43 nm is presented utilizing steady-state and time-resolved fluorometry. Special emphasis was dedicated to the absolute quantification of size- and environment-induced quenching of upconversion luminescence (UCL) by high-energy O–H and C–H vibrations from solvent and ligand molecules at different excitation power densities (P). In this context, the still-debated population pathways of the 4F9/2 energy level of Er3+ were examined. Our results highlight the potential of particle size and P value for color tuning based on the pronounced near-infrared emission of 12 nm UCNPs, which outweighs the red Er3+ emission under “strongly quenched” conditions and accounts for over 50% of total UCL in water. Because current rate equation models do not include such emissions, the suitability of these models for accurately simulating all (de)population pathways of small UCNPs must be critically assessed. Furthermore, we postulate population pathways for the 4F9/2 energy level of Er3+, which correlate with the size-, environment-, and P-dependent quenching states of the higher Er3+ energy levels.

Single-particle luminescence microscopy is a powerful method to extract information on biological systems that is not accessible by ensemble-level methods. Upconversion nanoparticles (UCNPs) are a particularly promising luminophore for single-particle microscopy as they provide stable, non-blinking luminescence and allow the avoidance of biological autofluorescence by their anti-Stokes emission. Recently, ensemble measurements of diluted aqueous dispersions of UCNPs have shown the instability of luminescence over time due to particle dissolution-related effects. This can be especially detrimental for single-particle experiments. However, this effect has never been estimated at the individual particle level. Here, the luminescence response of individual UCNPs under aqueous conditions is investigated by quantitative wide-field microscopy. The particles exhibit a rapid luminescence loss, accompanied by large changes in spectral response, leading to a considerable heterogeneity in their luminescence and band intensity ratio. Moreover, the dissolution-caused intensity loss is not correlated with the initial particle intensity or band ratio, which makes it virtually unpredictable. These effects and the subsequent development of their heterogeneity can be largely slowed down by adding millimolar concentrations of sodium fluoride in buffer. As a consequence, the presented data indicate that microscopy experiments employing UCNPs in an aqueous environment should be performed under conditions that carefully prevent these effects.

The authors describe the preparation of Eu3+-doped GdVO4 nanocrystals (NCs) by precipitation of the Cd3+(Eu3+)-citrate complex which was then converted to the respective vanadate by dialysis. The fractions of Eu3+ ranged from 5 to 100 mol%. The NCs were characterized by XRD, TEM, ICP-OES and dynamic light scattering which revealed that they possess superior colloidal stability in aqueous solutions in that no precipitation can be observed even after several months. The NCs display red and largely red shifted fluorescence (peaking at 618 nm) on photoexcitation at around 300 nm. Fluorescence is strongly quenched by hydrogen peroxide. It is also shown that the fraction of doping with Eu3+ strongly affects quenchability. Most efficient quenching by H2O2 is observed if the NCs are doped with 50% of Eu3+. The findings were exploited to develop a fluorometric assay for H2O2 that works in the 5 to 250 mu M concentration range, with a limit of detection as low as 1.6 mu M (at a signal-to-noise ratio of 3). The probe was further employed to design a highly sensitive enzymatic assay for glucose via measurement of the quantity of H2O2 formed as a result of the catalytic action of glucose oxidase. (C) 2016 Elsevier B.V. All rights reserved.

An upconversion laser scanner has been optimized to exploit the advantages of photon-upconverting nanoparticles (UCNPs) for background-free imaging on a macroscopic scale. A collimated 980 nm laser beam afforded high local excitation densities to account for the nonlinear luminescence response of UCNPs. As few as 2000 nanoparticles were detectable, and the linear dynamic range covered more than 5 orders of magnitude, which is essentially impossible by using conventional fluorescent dyes. UCNPs covered by a dye-doped silica shell were separated by agarose gel electrophoresis and scanned by a conventional fluorescence scanner as well as the upconversion scanner. Both optical labels could be detected independently. Finally, upconversion images of lateral flow test strips were recorded to facilitate the sensitive and quantitative detection of disease markers. A marker for the parasitic worm Schistosoma was used in this study.

We present a systematic study on the effect of surface ligands on the luminescence properties and colloidal stability of β-NaYF4:Yb3+,Er3+ upconversion nanoparticles (UCNPs), comparing nine different surface coatings to render these UCNPs water-dispersible and bioconjugatable. A prerequisite for this study was a large-scale synthetic method that yields ∼2 g per batch of monodisperse oleate-capped UCNPs providing identical core particles. These ∼23 nm sized UCNPs display an upconversion quantum yield of ∼0.35% when dispersed in cyclohexane and excited with a power density of 150 W cm−2, underlining their high quality. A comparison of the colloidal stability and luminescence properties of these UCNPs, subsequently surface modified with ligand exchange or encapsulation protocols, revealed that the ratio of the green (545 nm) and red (658 nm) emission bands determined at a constant excitation power density clearly depends on the surface chemistry. Modifications relying on the deposition of additional (amphiphilic) layer coatings, where the initial oleate coating is retained, show reduced non-radiative quenching by water as compared to UCNPs that are rendered water-dispersible via ligand exchange. Moreover, we could demonstrate that the brightness of the upconversion luminescence of the UCNPs is strongly affected by the type of surface modification, i.e., ligand exchange or encapsulation, yet hardly by the chemical nature of the ligand., Open Access Komponente aus der Allianzlizenz

Upconverting nanoparticles (UCNPs) are luminophores that have been investigated for a multitude of biological applications, notably low-background imaging, high-sensitivity assays, and cancer theranostics. In these applications, they are frequently used as a donor in resonance energy transfer (RET) pairs. However, because of the peculiarity and non-linearity of their luminescence mechanism, their behavior as a RET pair component has been difficult to predict quantitatively, preventing their optimization for subsequent applications. In this article, we assembled UCNP–organic dye RET systems and investigated their luminescence decays and spectra, with varying UCNP sizes and quantities of dyes grafted onto their surface. We observed an increase in RET efficiency with lower particle sizes and higher dye decoration. We also observed several unexpected effects, notably a quenching of UCNP luminescence bands that are not resonant with the absorption of organic dyes. We proposed a semi-empirical Monte Carlo model for predicting the behavior of UCNP-organic dye systems, and validated it by comparison with our experimental data. These findings will be useful for the development of more accurate UCNP-based assays, sensors, and imaging agents, as well as for optimization of UCNP–organic dye RET systems employed in cancer treatment and theranostics.

Upconversion nanoparticles (UCNPs) are attractive candidates for energy transfer-based analytical applications. In contrast to classical donor-acceptor pairs, these particles contain many emitting lanthanide ions together with numerous acceptor dye molecules at different distances to each other, strongly depending on the particle diameter. UCNPs with precisely controlled sizes between 10 and 43 nm were prepared and functionalized with rose bengal and sulforhodamine B by a ligand-exchange procedure. Time-resolved studies of the upconversion luminescence of the UCNP donor revealed a considerable shortening of the donor lifetime as a clear hint for Förster resonance energy transfer (FRET). FRET was most pronounced for 21 nm-sized UCNPs, yielding a FRET efficiency of 60%. At larger surface-to-volume ratios, the FRET efficiency decreased by an increasing competition of nonradiative surface deactivation. Such dye-UCNP architectures can also provide an elegant way to shift the UCNP emission color, since the fluorescence intensity of the organic dyes excited by FRET was comparable to that of the upconversion emission of smaller particles.

Upconverting luminescent nanoparticles (UCNPs) represent an interesting class of nanomaterials for bioanalytical applications. Due to their excitation in the near infrared region of the spectra, no fluorescence of biological compounds is trigged. Compared to other nanomaterials like quantum dots they exhibit low cytotoxicity, high photostability, no blinking and chemical inertness. Nevertheless, UCNPs suffer from low quantum efficiency. Here we report on two different core-shell particle systems which have a core consisting of NaYF4 doped with Yb3+/ Tm3+ and an additional inert shell (NaYF4) or an active shell (NaYF4 doped with Yb3+/Nd3+). Nanoparticles without Yb3+ as sensitizer can be excited at 980 nm. However, water has an absorption band in this region. This results in a reduction of the upconversion efficiency in aqueous systems and a heating of the solution. For bioanalytical application, more beneficial is the shifting of the wavelength to 808 nm by additional doping of the shell with Nd3+. Both core-shell systems were investigated in respect to the monitor enzymatic reactions of dehydrogenases and oxidases involving the generation of either NADH or FADH2.

Upconversion nanoparticles (UCNPs)/nanomaterials have the unique property to absorb near-infrared (NIR) light which leads to an anti-Stokes emission from the ultraviolet (UV) over the visible to the NIR range (Zhang 2014). The NIR light is absorbed by sensitizer ions (e.g., Yb3+ and Nd3+), and then transferred to so-called activator ions (e.g., Tm3+, Er3+, and Ho3+) via a nonradiative, resonant energy transfer process. The lanthanide ions are preferably embedded in host lattices with low phonon energy and similar ionic radii to reduce nonradiative deactivation processes (Haase and Schafer 2011; Suyver et al. 2006; Wang and Liu 2009). Fluorides combine low phonon energies (~300-400 cm−1) with high chemical stability compared to other halide ions or oxide materials. The doping ratio of the sensitizer and activator ions is very critical, in order to generate highly uorescent nanomaterials and to avoid self-quenching of the excited states. A NaYF4 hexagonal host latticeCONTENTS4.1 Introduction 69 4.2 Synthesis of UCNPs 71 4.3 Surface Modications of Hydrophobic UCNPs 72

CONSPECTUS: Photon upconversion nanoparticles (UCNPs) have emerged as a promising new class of nanomaterials due to their ability to convert near-IR light into visible luminescence. Unfortunately, most efficient methods for preparing UCNPs yield hydrophobic materials, but water-dispersibility is needed in the major fields of applications of UCNPs, that is, in bioimaging, labeling, and bioassays. Numerous methods therefore have been reported in the past years to convert the hydrophobic surface of UCNPs to a more hydrophilic one so to render them dispersible in aqueous systems. We present a classification respective for these strategies and assess the main methods. These include (A) chemical modification of the hydrophobic (typically oleate) ligand on the surface, (B) addition of an extra layer, (C) addition of a thin shell on top of the UCNP, and (D) complete replacement of the original ligand by another one. Chemical modification (A) involves oxidation of the oleate or oleylamine and leads to particles with terminal oxygen functions. This method is less often used because solutions of the resulting UCNPs in water have limited colloidal stability, protocols are time-consuming and often give low yields, and only a limited number of functional groups can be introduced. Methods B and C involve coating of UCNPs with amphiphiles or with shells made from silica oxide, titanium oxide, or metallic gold or silver. These methods are quite versatile in terms of further modifications, for example, by further cross-linking or by applying thiol-gold chemistry. Growing an extra shell is, however, often accompanied by a higher polydispersity. Method D can be divided into subgroups based on either (i) the direct (single-step) replacement of the native ligand by a new ligand or (ii) two-step protocols using nitrosyltetrafluoroborate (NOBF4) or strong acids as reagents to produce ligand-free UCNPs prior to the attachment of a new ligand. These methods are simple and versatile, and the distance between the new ligand and the luminescent particle can be well controlled. However, the particles often have limited stability in buffer systems. The methods described also are of wider interest because they are likely to be applicable to other kinds of nanomaterials. We additionally address the need for (a) a better control of particle size and homogeneity during synthesis, (b) more reproducible methods for surface loading and modification, (c) synthetic methods giving higher yields of UCNPs, (d) materials displaying higher quantum yields in water solution without the need for tedious surface modifications, (e) improved methods for workup (including the suppression of aggregation), (f) new methods for surface characterization, and (g) more affordable reagents for use in surface modification. It is noted that most synthetic research in the area is of the trial-and-error kind, presumably due to the lack of understanding of the mechanisms causing current limitations. Finally, all particles are discussed in terms of their biocompatibility (as far as data are available), which is quintessential in terms of imaging, the largest field of application.