Virginia Altoe

Non-blinking and photostable upconverted luminescence from single lanthanide-doped nanocrystals

The development of probes for single-molecule imaging has dramatically facilitated the study of individual molecules in cells and other complex environments. Single-molecule probes ideally exhibit good brightness, uninterrupted emission, resistance to photobleaching, and minimal spectral overlap with cellular autofluorescence. However, most single-molecule probes are imperfect in several of these aspects, and none have been shown to possess all of these characteristics. Here we show that individual lanthanide-doped upconverting nanoparticles (UCNPs)—specifically, hexagonal phase NaYF4 (β-NaYF4) nanocrystals with multiple Yb3+ and Er3+ dopants—emit bright anti-Stokes visible upconverted luminescence with exceptional photostability when excited by a 980-nm continuous wave laser. Individual UCNPs exhibit no on/off emission behavior, or “blinking,” down to the millisecond timescale, and no loss of intensity following an hour of continuous excitation. Amphiphilic polymer coatings permit the transfer of hydrophobic UCNPs into water, resulting in individual water-soluble nanoparticles with undiminished photophysical characteristics. These UCNPs are endocytosed by cells and show strong upconverted luminescence, with no measurable anti-Stokes background autofluorescence, suggesting that UCNPs are ideally suited for single-molecule imaging experiments.

Evolution of Structure and Chemistry of Bimetallic Nanoparticle Catalysts under Reaction Conditions

Three series of bimetallic nanoparticle catalysts (Rh(x)Pd(1-x), Rh(x)Pt(1-x), and Pd(x)Pt(1-x), x = 0.2, 0.5, 0.8) were synthesized using one-step colloidal chemistry. X-ray photoelectron spectroscopy (XPS) depth profiles using different X-ray energies and scanning transmission electron microscopy showed that the as-synthesized Rh(x)Pd(1-x) and Pd(x)Pt(1-x) nanoparticles have a core-shell structure whereas the Rh(x)Pt(1-x) alloys are more homogeneous in structure. The evolution of their structures and chemistry under oxidizing and reducing conditions was studied with ambient-pressure XPS (AP-XPS) in the Torr pressure range. The Rh(x)Pd(1-x) and Rh(x)Pt(1-x) nanoparticles undergo reversible changes of surface composition and chemical state when the reactant gases change from oxidizing (NO or O(2) at 300 degrees C) to reducing (H(2) or CO at 300 degrees C) or catalytic (mixture of NO and CO at 300 degrees C). In contrast, no significant change in the distribution of the Pd and Pt atoms in the Pd(x)Pt(1-x) nanoparticles was observed. The difference in restructuring behavior under these reaction conditions in the three series of bimetallic nanoparticle catalysts is correlated with the surface free energy of the metals and the heat of formation of the metallic oxides. The observation of structural evolution of bimetallic nanoparticles under different reaction conditions suggests the importance of in situ studies of surface structures of nanoparticle catalysts.

Transfer-Free Batch Fabrication of Large-Area Suspended Graphene Membranes

We demonstrate a process for batch production of large-area (100-3000 microm(2)) patterned free-standing graphene membranes on Cu scaffolds using chemical vapor deposition (CVD)-grown graphene. This technique avoids the use of silicon and transfers of graphene. As one application of this technique, we fabricate transmission electron microscopy (TEM) sample supports. TEM characterization of the graphene membranes reveals relatively clean, highly TEM-transparent, single-layer graphene regions ( approximately 50% by area) and, despite the polycrystalline nature of CVD graphene, membrane yields as high as 75-100%. This high yield verifies that the intrinsic strength and integrity of CVD-grown graphene films is sufficient for sub-100 microm width membrane applications. Elemental analysis (electron energy loss spectroscopy (EELS) and X-ray energy-dispersive spectroscopy (EDS)) of the graphene membranes reveals some nanoscaled contamination left over from the etching process, and we suggest several ways to reduce this contamination and improve the quality of the graphene for electronic device applications. This large-scale production of suspended graphene membranes facilitates access to the two-dimensional physics of graphene that are suppressed by substrate interactions and enables the widespread use of graphene-based sample supports for electron and optical microscopy.

Structure vs. properties — chirality, optics and shapes — in amphiphilic porphyrin J-aggregates

The structure of the meso-tetrakis(4-sulfonatophenyl)porphyrin (TPPS4) J-aggregates could be determined by X-ray and electron diffraction methods. A sheet-like architecture reveals the relationship between structure and chirality, optics and shapes of the J-aggregates of the meso 4-sulfonatophenyl- and phenyl-substituted porphyrins. The structure of the J-aggregates of H4TPPS4 belongs to the chiral space group P21 and includes four porphyrin molecules in its unit cell. The intermolecular stabilization of the zwitterionic units by hydrogen bonding and electrostatic interactions between the positively charged central NH groups and the periphery anionic sulfonato groups results in a structure of porphyrins sheets along the [01] plane direction. The structure of the sheet on the [01] plane is already chiral and its molecular architecture explains the simultaneous presence of H- and J-aggregate bands in their absorption spectra. This structure also accounts for the high similarity observed between the absorption spectra of different mesomorphs of the same substance and even between different members of the series of meso-4-sulfonatophenyl- and aryl-substituted diprotonated porphyrins. The possibility, or not, of the sheet-like structure on [01] to interact with other layers, either through ionic or hydrophobic interactions, depends on the substitution pattern at the meso-positions of the porphyrin ring. Thus, the different morphologies of the particles [mono- bi- and multilayered] of this series of J-aggregates are explained taking into account the role that the fourth meso-substituent plays in the interlayer stabilization. The results suggest that supramolecular helicity, previously detected in several J-aggregates, is not the explanation of their chirality but would be the expression of the intrinsic chirality of the packing between building blocks.

Electrical Transport Properties of Oligothiophene-Based Molecular Films Studied by Current Sensing Atomic Force Microscopy

Using conducting probe atomic force microscopy (CAFM) we have investigated the electrical conduction properties of monolayer films of a pentathiophene derivative on a SiO(2)/Si-p+ substrate. By a combination of current-voltage spectroscopy and current imaging we show that lateral charge transport takes place in the plane of the monolayer via hole injection into the highest occupied molecular orbitals of the pentathiophene unit. Our CAFM data suggest that the conductivity is anisotropic relative to the crystalline directions of the molecular lattice.

The plastic nature of the human bone–periodontal ligament–tooth fibrous joint

This study investigates bony protrusions within a narrowed periodontal ligament space (PDL-space) of a human bone-PDL-tooth fibrous joint by mapping structural, biochemical, and mechanical heterogeneity. Higher resolution structural characterization was achieved via complementary atomic force microscopy (AFM), nano-transmission X-ray microscopy (nano-TXM), and microtomography (MicroXCT™). Structural heterogeneity was correlated to biochemical and elemental composition, illustrated via histochemistry and microprobe X-ray fluorescence analysis (μ-XRF), and mechanical heterogeneity evaluated by AFM-based nanoindentation. Results demonstrated that the narrowed PDL-space was due to invasion of bundle bone (BB) into PDL-space. Protruded BB had a wider range with higher elastic modulus values (2-8GPa) compared to lamellar bone (0.8-6GPa), and increased quantities of Ca, P and Zn as revealed by μ-XRF. Interestingly, the hygroscopic 10-30μm interface between protruded BB and lamellar bone exhibited higher X-ray attenuation similar to cement lines and lamellae within bone. Localization of the small leucine rich proteoglycan biglycan (BGN) responsible for mineralization was observed at the PDL-bone interface and around the osteocyte lacunae. Based on these results, it can be argued that the LB-BB interface was the original site of PDL attachment, and that the genesis of protruded BB identified as protrusions occurred as a result of shift in strain. We emphasize the importance of bony protrusions within the context of organ function and that additional study is warranted.

Electron microscopy reveals structure and morphology of one molecule thin organic films.

Transmission electron microscopy was used to determine the structure of molecular films of self-assembled monolayers of pentathiophene derivatives supported on various electron transparent substrates. Despite the extreme beam sensitivity of the monolayers, structural crystallographic maps were obtained that revealed the nanoscale structure of the film. The image resolution is determined by the minimum beam diameter that the radiation hardness of the monolayer can support, which in our case is about 90 nm for a beam current of 5 × 10(6) e(-)/s. Electron diffraction patterns were collected while scanning a parallel electron beam over the film. These maps contain uncompromised information of the size, symmetry and orientation of the unit cell, orientation and structure of the domains, degree of crystallinity, and their variation on the micrometer scale, which are crucial to understand the electrical transport properties of the organic films. This information allowed us to track small changes in the unit cell size driven by the chemical modification of the support film.

Elastic discontinuity due to ectopic calcification in a human fibrous joint

Disease can alter natural ramp-like elastic gradients to steeper step-like profiles at soft-hard tissue interfaces. Prolonged function can further mediate mechanochemical events that alter biomechanical response within diseased organs. In this study, a human bone-tooth fibrous joint was chosen as a model system, in which the effects of bacterial-induced disease, i.e. periodontitis, on natural elastic gradients were investigated. Specifically, the effects of ectopic biomineral, i.e. calculus, on innate chemical and elastic gradients within the cementum-dentin complex, both of which are fundamental parameters to load-bearing tissues, are investigated through comparisons with a healthy complex. Complementary techniques for mapping changes in physicochemical properties as a result of disease included micro X-ray computed tomography, microprobe micro X-ray fluorescence imaging, transmission electron and atomic force microscopy (AFM) techniques, and AFM-based nanoindentation. Results demonstrated primary effects as derivatives of ectopic mineralization within the diseased fibrous joint. Ectopic mineralization with no cementum resorption, but altered cementum physicochemical properties with increasing X-ray attenuation, exhibited stratified concretion with increasing X-ray fluorescence counts of calcium and phosphorus elements in the extracellular matrix in correlation with decreased hygroscopicity, indenter displacement, and apparent strain-relieving characteristics. Disease progression, identified as concretion through the periodontal ligament (PDL)-cementum enthesis, and sometimes the originally hygroscopic cementum-dentin junction, resulted in a significantly increased indentation elastic modulus (3.16±1.19 GPa) and a shift towards a discontinuous interface compared with healthy conditions (1.54±0.83 GPa) (Students t-test, P<0.05). The observed primary effects could result in secondary downstream effects, such as compromised mechanobiology at the mechanically active PDL-cementum enthesis that can catalyze progression of disease.

Effect of proteoglycans at interfaces as related to location, architecture, and mechanical cues.

INTRODUCTION Covalently bound functional GAGs orchestrate tissue mechanics through time-dependent characteristics. OBJECTIVE The role of specific glycosaminoglycans (GAGs) at the ligament-cementum and cementum-dentin interfaces within a human periodontal complex were examined. Matrix swelling and resistance to compression under health and modeled diseased states was investigated. MATERIALS AND METHODS The presence of keratin sulfate (KS) and chondroitin sulfate (CS) GAGs at the ligament-cementum and cementum-dentin interfaces in human molars (N=5) was illustrated by using enzymes, atomic force microscopy (AFM), and AFM-based nanoindentation. The change in physical characteristics of modeled diseased states through sequential digestion of keratin sulfate (KS) and chondroitin sulfate (CS) GAGs was investigated. One-way ANOVA tests with P<0.05 were performed to determine significant differences between groups. Additionally, the presence of mineral within the seemingly hygroscopic interfaces was investigated using transmission electron microscopy. RESULTS Immunohistochemistry (N=3) indicated presence of biglycan and fibromodulin small leucine rich proteoglycans at the interfaces. Digestion of matrices with enzymes confirmed the presence of KS and CS GAGs at the interfaces by illustrating a change in tissue architecture and mechanics. A significant increase in height (nm), decrease in elastic modulus (GPa), and tissue deformation rate (nm/s) of the PDL-C attachment site (215±63-424±94nm; 1.5±0.7-0.4±0.2GPa; 21±7-48±22nm/s), and cementum-dentin interface (122±69-360±159nm; 2.9±1.3-0.7±0.3GPa; 18±4-30±6nm/s) was observed. CONCLUSIONS The sequential removal of GAGs indicated loss in intricate structural hierarchy of hygroscopic interfaces. From a mechanics perspective, GAGs provide tissue recovery/resilience. The results of this study provide insights into the role of GAGs toward conserved tooth movement in the socket in response to mechanical loads, and modulation of potentially deleterious strain at tissue interfaces.

A Force on the Crown and Tug of War in the Periodontal Complex

The load-bearing dentoalveolar fibrous joint is composed of biomechanically active periodontal ligament (PDL), bone, cementum, and the synergistic entheses of PDL-bone and PDL-cementum. Physiologic and pathologic loads on the dentoalveolar fibrous joint prompt natural shifts in strain gradients within mineralized and fibrous tissues and trigger a cascade of biochemical events within the widened and narrowed sites of the periodontal complex. This review highlights data from in situ biomechanical simulations that provide tooth movements relative to the alveolar socket. The methods and subsequent results provide a reasonable approximation of strain-regulated biochemical events resulting in mesial mineral formation and distal resorption events within microanatomical regions at the ligament-tethered/enthesial ends. These biochemical events, including expressions of biglycan, decorin, chondroitin sulfated neuroglial 2, osteopontin, and bone sialoprotein and localization of various hypertrophic progenitors, are observed at the alkaline phosphatase–positive widened site, resulting in mineral formation and osteoid/cementoid layers. On the narrowed side, tartrate-resistant acid phosphatase regions can lead to a sequence of clastic activities resulting in resorption pits in bone and cementum. These strain-regulated biochemical and subsequently biomineralization events in the load-bearing periodontal complex are critical for maintenance of the periodontal space and overall macroscale joint biomechanics.