Nanocrystals of β-NaYF4:Yb3+, Er3+ generally have lower NIR-to-visible upconversion (UC) internal quantum efficiency, IQE, compared to high-quality bulk materials, and exhibit more rapid UC dynamics, typical of quenching, when excited with a pulsed source near 980 nm. The addition of a protective shell increases the IQE of the nanocrystals and slows the overall excited-state dynamics. Here, we show that an extension of a recently developed model for UC in powders of micron-sized β-NaYF4:18%Yb3+, 2%Er3+ crystals correctly predicts the time-resolved luminescence curve shapes, relative intensities, and observed drop in IQE of the various emission lines for core and core–shell nanoparticles following pulsed excitation. The model clearly shows that the nanoscale effect on visible upconversion luminescence in these materials, with typical high-Yb3+ and low-Er3+ doping, is largely due to rapid energy migration among Yb3+(2F5/2) and Er3+(4I11/2) ions at the 1 μm energy level, such that an equilibrium is achieved between interior sites and rapidly relaxing surface sites. The faster kinetics observed in visible emission following pulsed NIR excitation is mainly a propagation of the effect of surface quenching of the 1 μm reservoir states and is not due to direct quenching of the visible emitting states themselves. For Er3+ ions contributing to UC emission, the relaxation rate constants for the blue (2H9/2), green (2H11/2, 4S3/2), and red (4F9/2) emitting states are essentially unchanged from their bulk values, indicating that Er3+ ions close to the nanoparticle surface are nearly silent with regard to UC. The addition of a passive β-NaYF4 shell retards the drain of the 1 μm excitation reservoir and recovers the participation of outer Er3+ sites in UC. The dependence of IQE on shell thickness is well explained in terms of a Förster-type model describing an energy donor (Er3+, Yb3+) interacting with a thin plane layer of acceptors (oleate). The UC behavior of both the core and the core–shell nanocrystals can be modeled, almost quantitatively, solely on the basis of quenching at the 1 μm level, without separate consideration of a near-surface Er3+ population. However, a two-layer model for the core nanoparticles is revealing with regard to the modest extent to which near-surface ions do participate in UC and gives a better representation of the detailed dynamics of the NIR emitting states. A method is presented for allowing investigators to estimate the IQE for any nanosample (with 18% Yb3+, 2%Er3+ doping) as a function of excitation power density (cw) or pulse-energy density based on the low pulse energy measurement of the decay constant for the 1 μm emission.

Clathrin-mediated endocytosis (CME) internalizes plasma membrane by reshaping small regions of the cell surface into spherical vesicles. The key mechanistic question of how coat assembly produces membrane curvature has been studied with molecular and cellular structural biology approaches, without direct visualization of the process in living cells; resulting in two competing models for membrane bending. Here we use polarized total internal reflection fluorescence microscopy (pol-TIRF) combined with electron, atomic force, and super-resolution optical microscopy to measure membrane curvature during CME. Surprisingly, coat assembly accommodates membrane bending concurrent with or after the assembly of the clathrin lattice. Once curvature began, CME proceeded to scission with robust timing. Four color pol-TIRF showed that CALM accumulated at high levels during membrane bending, implicating its auxiliary role in curvature generation. We conclude that clathrin-coat assembly is versatile and that multiple membrane-bending trajectories likely reflect the energetics of coat assembly relative to competing forces.

Spectroscopic imaging and time-resolved spectroscopy are used to study the surface plasmon polariton (SPP) enhanced infrared to visible upconversion luminescence from NaYF 4 :Tm:Yb nanoparticles embedded in polymethyl methylacrylate (PMMA) supported on Au nanopillar arrays. The arrays have a lattice resonance associated with the SPP near 980 nm, near-resonant with the peak absorption of the Yb 3+ ion, while a local surface plasmon resonance (LSPR) associated with the individual pillars is seen to enhance the near-infrared emission of Tm 3+ ions near 780 nm. The two combined channels of enhancement result in a significantly higher enhancement of the near-infrared emission when compared to the visible upconversion lines of the Tm 3+ ion, consistent with the interpretation of sequential surface plasmon assisted absorption and emission at two separate and disparate energies. The presence of SPP and LSPR was confirmed by spectrally resolved reflectivity, and the mechanisms for luminescence enhancement were further confirmed by time-resolved measurements of the upconversion luminescence.

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Summary ParagraphClathrin-mediated endocytosis internalizes membrane from the cell surface by reshaping flat regions of membrane into spherical vesicles(1, 2). The relationship between membrane bending and clathrin coatomer assembly has been inferred from electron microscopy and structural biology, without directly visualization of membrane bending dynamics (3–6). This has resulted in two distinct and opposing models for how clathrin bends membrane (7–10). Here, polarized Total Internal Reflection Fluorescence microscopy was improved and combined with electron microscopy, atomic force microscopy, and super-resolution imaging to measure membrane bending during endogenous clathrin and dynamin assembly in living cells. Surprisingly, and not predicted by either model, the timing of membrane bending was variable relative to clathrin assembly. Approximately half of the time, membrane bending occurs at the start of clathrin assembly, in the other half, the onset of membrane bending lags clathrin arrival, and occasionally completely assembled flat clathrin transitions into a pit. Importantly, once the membrane bends, the process proceeds to scission with similar timing. We conclude that the pathway of coatomer formation is versatile and can bend the membrane during or after the assembly of the clathrin lattice. These results highlight the heterogeneity in this fundamental biological process, and provide a more complete nanoscale view of membrane bending dynamics during endocytosis.