Aiming at the derivation of a generalized procedure for the straightforward preparation of particles fluorescing in the visible and near-infrared (NIR) spectral region, different swelling procedures ...for the loading of the hydrophobic polarity-probe Nile Red into nano- and micrometer sized polystyrene particles were studied and compared with respect to the optical properties of the resulting particles. The effect of the amount of incorporated dye on the spectroscopic properties of the particles was investigated for differently sized beads with different surface chemistries, i.e., non-functionalized, amino-modified and PEG-grafted surfaces. Moreover, photostability and leaking studies were performed. The main criterion for the optimization of the dye loading procedures was a high and thermally and photochemically stable fluorescence output of the particles for the future application of these systems as fluorescent labels.
Luminescence techniques are among the most widely used detection methods in the life and material sciences. At the core of these methods is an ever-increasing variety of fluorescent reporters (i.e., ...simple dyes, fluorescent labels, probes, sensors and switches) from different fluorophore classes ranging from small organic dyes and metal ion complexes, quantum dots and upconversion nanocrystals to differently sized fluorophore-doped or fluorophore-labeled polymeric particles. A key parameter for fluorophore comparison is the fluorescence quantum yield (Φf), which is the direct measure for the efficiency of the conversion of absorbed light into emitted light. In this protocol, we describe procedures for relative and absolute determinations of Φf values of fluorophores in transparent solution using optical methods, and we address typical sources of uncertainty and fluorophore class-specific challenges. For relative determinations of Φf, the sample is analyzed using a conventional fluorescence spectrometer. For absolute determinations of Φf, a calibrated stand-alone integrating sphere setup is used. To reduce standard-related uncertainties for relative measurements, we introduce a series of eight candidate quantum yield standards for the wavelength region of ∼350-950 nm, which we have assessed with commercial and custom-designed instrumentation. With these protocols and standards, uncertainties of 5-10% can be achieved within 2 h.
In order to assess the anisotropy-related uncertainties of relatively
determined photoluminescence quantum yields (
) of
molecular emitters and luminescent nanomaterials, we compared
values measured ...without and with polarizers using
magic angle conditions and studied systematically the dependence of
the detected emission intensity on the polarizer settings for samples
of varying anisotropy. This includes a dispersion of a spherical
quantum dot (QD) with an ideally isotropic emission, a solution of
a common small organic dye in a fluid solvent as well as dispersions
of elongated quantum dot rods (QDR) with an anisotropic luminescence
and a small organic dye in a rigid polymeric matrix, as ideally
anisotropic emitter. Our results show that for instruments lacking
polarizers, anisotropy-related measurement uncertainties of relative
photoluminescence quantum yields can amount to more than 40%,
with the size of these systematic errors depending on the difference
in emission anisotropy between the sample and the standard.
The commercial availability of stand-alone setups for the determination of absolute photoluminescence quantum yields (Φf) in conjunction with the increasing use of integrating sphere accessories for ...spectrofluorometers is expected to have a considerable influence not only on the characterization of chromophore systems for use in optical and opto-electronic devices, but also on the determination of this key parameter for (bio)analytically relevant dyes and functional luminophores. Despite the huge potential of systems measuring absolute Φf values and the renewed interest in dependable data, evaluated protocols for even the most elementary case, the determination of the fluorescence quantum yield of transparent dilute solutions of small organic dyes with integrating sphere methods, are still missing. This encouraged us to evaluate the performance and sources of uncertainty of a simple commercial integrating sphere setup with dilute solutions of two of the best characterized fluorescence quantum yield standards, quinine sulfate dihydrate and rhodamine 101, strongly differing in spectral overlap between absorption and emission. Special attention is dedicated to illustrate common pitfalls of this approach, thereby deriving simple procedures to minimize measurement uncertainties and improve the comparability of data for the broad community of users of fluorescence techniques.
The rational design of nano- and micrometer-sized particles with tailor-made optical properties for biological, diagnostic, and photonic applications requires tools to characterize the ...signal-relevant properties of these typically scattering bead suspensions. This includes methods for the preferably nondestructive quantification of the number of fluorophores per particle and the measurement of absolute fluorescence quantum yields and absorption coefficients of suspensions of fluorescent beads for material performance optimization and comparison. Here, as a first proof-of-concept, we present the first time determination of the number of dye molecules per bead using nondestructive quantitative (19F) NMR spectroscopy and 1000 nm-sized carboxylated polystyrene particles loaded with varying concentrations of the laser dye coumarin 153 containing a CF3 group. Additionally, the signal-relevant optical properties of these dye-loaded particles were determined in aqueous suspension in comparison to the free dye in solvents of different polarity with a custom-built integrating sphere setup that enables spectrally resolved measurements of emission, transmission, and reflectance as well absolute fluorescence quantum yields. These measurements present an important step toward absolute brightness values and quantitative fluorescence analysis with particle systems that can be exploited, for example, for optical imaging techniques and different fluorescence assays as well as for the metrological traceability of fluorescence methods.
Upconversion core/shell nanocrystals with different mean sizes ranging from 15 to 45 nm were prepared via a modified synthesis procedure based on anhydrous rare‐earth acetates. All particles consist ...of a core of NaYF4:Yb,Er, doped with 18 % Yb3+ and 2 % Er3+, and an inert shell of NaYF4, with the shell thickness being equal to the radius of the core particle. Absolute measurements of the photoluminescence quantum yield at a series of different excitation power densities show that the quantum yield of 45 nm core/shell particles is already very close to the quantum yield of microcrystalline upconversion phosphor powder. Smaller core/shell particles prepared by the same method show only a moderate decrease in quantum yield. The quantum yield of 15 nm core/shell particles, for instance, is reduced by a factor of three compared to the bulk upconversion phosphor at high power densities (100 W cm−2) and by approximately a factor of 10 at low power densities (1 W cm−2).
A modified synthesis based on anhydrous rare‐earth acetates gives upconversion core/shell nanocrystals with different mean sizes ranging from 15 to 45 nm. Absolute measurements of the photoluminescence quantum yield show that the quantum yield of 45 nm core/shell particles is close to that of microcrystalline upconversion phosphor powder.
High sensitizer and activator concentrations have been increasingly examined to improve the performance of multi-color emissive upconversion (UC) nanocrystals (UCNC) like NaYF
4
:Yb,Er and first ...strategies were reported to reduce concentration quenching in highly doped UCNC. UC luminescence (UCL) is, however, controlled not only by dopant concentration, yet by an interplay of different parameters including size, crystal and shell quality, and excitation power density (
P
). Thus, identifying optimum dopant concentrations requires systematic studies of UCNC designed to minimize additional quenching pathways and quantitative spectroscopy. Here, we quantify the dopant concentration dependence of the UCL quantum yield (
Φ
UC
) of solid NaYF
4
:Yb,Er/NaYF
4
:Lu upconversion core/shell nanocrystals of varying Yb
3+
and Er
3+
concentrations (Yb
3+
series: 20%–98% Yb
3+
; 2% Er
3+
; Er
3+
series: 60% Yb
3+
; 2%–40% Er
3+
). To circumvent other luminescence quenching processes, an elaborate synthesis yielding OH-free UCNC with record
Φ
UC
of ∼9% and ∼25 nm core particles with a thick surface shell were used. High Yb
3+
concentrations barely reduce
Φ
UC
from ∼9% (20% Yb
3+
) to ∼7% (98% Yb
3+
) for an Er
3+
concentration of 2%, thereby allowing to strongly increase the particle absorption cross section and UCNC brightness. Although an increased Er
3+
concentration reduces
Φ
UC
from ∼7% (2% Er
3+
) to 1% (40%) for 60% Yb
3+
. Nevertheless, at very high
P
(> 1 MW/cm
2
) used for microscopic studies, highly Er
3+
-doped UCNC display a high brightness because of reduced saturation. These findings underline the importance of synthesis control and will pave the road to many fundamental studies of UC materials.
A systematic study of the luminescence properties of monodisperse β-NaYF
4
: 20% Yb
3+
, 2% Er
3+
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
4
F
9/2
energy level of Er
3+
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 Er
3+
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
4
F
9/2
energy level of Er
3+
, which correlate with the size-, environment-, and P-dependent quenching states of the higher Er
3+
energy levels.
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.
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