Huan-Cheng Chang

Mass production and dynamic imaging of fluorescent nanodiamonds.

Fluorescent nanodiamond is a new nanomaterial that possesses several useful properties, including good biocompatibility, excellent photostability and facile surface functionalizability. Moreover, when excited by a laser, defect centres within the nanodiamond emit photons that are capable of penetrating tissue, making them well suited for biological imaging applications. Here, we show that bright fluorescent nanodiamonds can be produced in large quantities by irradiating synthetic diamond nanocrystallites with helium ions. The fluorescence is sufficiently bright and stable to allow three-dimensional tracking of a single particle within the cell by means of either one- or two-photon-excited fluorescence microscopy. The excellent photophysical characteristics are maintained for particles as small as 25 nm, suggesting that fluorescent nanodiamond is an ideal probe for long-term tracking and imaging in vivo, with good temporal and spatial resolution.

In Vivo Imaging and Toxicity Assessments of Fluorescent Nanodiamonds in Caenorhabditis elegans

Nanoscale carbon materials hold great promise for biotechnological and biomedical applications. Fluorescent nanodiamond (FND) is a recent new addition to members of the nanocarbon family. Here, we report long-term in vivo imaging of FNDs in Caenorhabditis elegans (C. elegans) and explore the nano-biointeractions between this novel nanomaterial and the model organism. FNDs are introduced into wild-type C. elegans by either feeding them with colloidal FND solution or microinjecting FND suspension into the gonads of the worms. On feeding, bare FNDs stay in the intestinal lumen, while FNDs conjugated with biomolecules (such as dextran and bovine serum albumin) are absorbed into the intestinal cells. On microinjection, FNDs are dispersed in the gonad and delivered to the embryos and eventually into the hatched larvae in the next generation. The toxicity assessments, performed by employing longevity and reproductive potential as physiological indicators and measuring stress responses with use of reporter genes, show that FNDs are stable and nontoxic and do not cause any detectable stress to the worms. The high brightness, excellent photostability, and nontoxic nature of the nanomaterial have enabled continuous imaging of the whole digestive system and tracking of the cellular and developmental processes of the living organism for several days.

Tracking the engraftment and regenerative capabilities of transplanted lung stem cells using fluorescent nanodiamonds

Lung stem/progenitor cells are potentially useful for regenerative therapy, for example in repairing damaged or lost lung tissue in patients. Several optical imaging methods and probes have been used to track how stem cells incorporate and regenerate themselves in vivo over time. However, these approaches are limited by photobleaching, toxicity and interference from background tissue autofluorescence. Here we show that fluorescent nanodiamonds, in combination with fluorescence-activated cell sorting, fluorescence lifetime imaging microscopy and immunostaining, can identify transplanted CD45–CD54+CD157+ lung stem/progenitor cells in vivo, and track their engraftment and regenerative capabilities with single-cell resolution. Fluorescent nanodiamond labelling did not eliminate the cells’ properties of self-renewal and differentiation into type I and type II pneumocytes. Time-gated fluorescence imaging of tissue sections of naphthalene-injured mice indicates that the fluorescent nanodiamond-labelled lung stem/progenitor cells preferentially reside at terminal bronchioles of the lungs for 7 days after intravenous transplantation. Supplementary information The online version of this article (doi:10.1038/nnano.2013.147) contains supplementary material, which is available to authorized users.

Receptor‐Mediated Cellular Uptake of Folate‐Conjugated Fluorescent Nanodiamonds: A Combined Ensemble and Single‐Particle Study

Fluorescent nanodiamonds (FNDs) are nontoxic and photostable nanomaterials, ideal for long-term in vivo imaging applications. This paper reports that FNDs with a size of approximately 140 nm can be covalently conjugated with folic acid (FA) for receptor-mediated targeting of cancer cells at the single-particle level. The conjugation is made by using biocompatible polymers, such as polyethylene glycol, as crosslinked buffer layers. Ensemble-averaged measurements with flow cytometry indicate that more than 50% of the FA-conjugated FND particles can be internalized by the cells (such as HeLa cells) through receptor-mediated endocytosis, as confirmed by competitive inhibition assays. Confocal fluorescence microscopy reveals that these FND particles accumulate in the perinuclear region. The absolute number of FNDs internalized by HeLa cells after 3 h of incubation at a particle concentration of 10 microg mL(-1) is in the range of 100 particles per cell. The receptor-mediated uptake process is further elucidated by single-particle tracking of 35-nm FNDs in three dimensions and real time during the endocytosis.

The Exocytosis of Fluorescent Nanodiamond and Its Use as a Long-Term Cell Tracker

Fluorescent nanodiamond (FND) has excellent biocompatibility and photostability, making it well suited for long-term labeling and tracking of cancer and stem cells. To prove the concept, the exocytosis of FND particles (size ≈100 nm) from three cell lines--HeLa cervical cancer cells, 3T3-L1 pre-adipocytes, and 489-2.1 multipotent stromal cells--is studied in detail. FND labeling is performed by incubating the cells in a serum-free medium containing 80 μg mL(-1) FND for 4 h. No significant alteration in growth or proliferation of the FND-labeled cells, including the multipotent stromal cells, is observed for up to 8 days. Flow cytometric analysis, in combination with parallel cell doubling-time measurements, indicates that there is little (≈15% or less) excretion of the endocytosed FND particles after 6 days of labeling for both HeLa and 489-2.1 cells, but exocytosis occurs more readily (up to 30%) for 3T3-L1 preadipocytes. A comparative experiment with FND and the widely used dye, carboxyfluorescein diacetate succinimidyl ester, demonstrates that the nanoparticle platform is a promising alternate probe for long-term cell labeling and tracking applications.

Infrared spectroscopy of CO on NaCl(100) IV. Bandshape analysis

The infrared bandshape of monolayer CO on NaCl(100) at 4 K is slightly asymmetric and its bandwidth of 0.09 cm−1 is the narrowest observed for any surface molecule. From a variety of experiments we know that this band profile is determined by heterogeneous rather than homogeneous broadening. We offer an explanation for the experimentally observed bandshape by proposing that irregularities in the surface of the cleaved NaCl crystals give rise to many distinct two-dimensional domains of physisorbed CO. A first-order perturbation calculation, which generates the infrared cross-section as a function of domain size, has been used to estimate that on the order of 105 molecules reside in an average size domain. The accuracy of the calculation, whose Hamiltonian represents the intermolecular potential among CO molecules by electric multipole coupling, will be discussed. We use the same coupling Hamiltonian to explore the effect of submonolayer coverages, isotopic dilution, and surface heterogeneities on the infrared spectroscopy of CO on NaCl(100). Our calculations can account for some, but not all, of the observed spectroscopic data.

Sub-20-nm fluorescent nanodiamonds as photostable biolabels and fluorescence resonance energy transfer donors.

Adv. Mater. 2010, 22, 843–847 2010 WILEY-VCH Verlag Gm A current trend in fluorescent probe technology is to expand the role of fluorophores that emit light in the far red and near infrared region for biomedical use. The negatively charged nitrogenvacancy defect center, (N-V) , in type Ib diamond is one of such fluorophores and has drawn much attention in recent years. The center exhibits several distinct features such as extended red emission at 700 nm, near-unity fluorescence quantum yield, and ultrahigh photostability (neither photobleaching nor photoblinking). These characteristics, along with the diverse surface functionalizability and non-toxic nature of the material, have made fluorescent nanodiamond (FND) a promising candidate among other conventional markers and labels, for example, organic dyes, fluorescent proteins, and quantum dots, for bioimaging applications. Despite the unique photophysical and biochemical properties of FND, the size is an important parameter to characterize. Decreasing the size, clearly, will increase the mobility of the fluorescent nanoprobe inside a cell, avoid altering the properties of targeted molecules, and enhance its translocation into cell nuclei. Additionally, particles of size in the range of 10 nm are more suitable for biolabeling and fluorescence resonance energy transfer (FRET) applications. However, so far the sizecontrolled production of FNDs has not yet been so well developed as that of other probes and scale-up synthesis of smaller and brighter FNDs remains a challenging task. In this communication, we report procedures of production and isolation of sub-20-nm FND particles utilizing He ion irradiation and differential centrifugation methods, and experiments demonstrating that they are useful as far-red fluorescent biolabels as well as FRET donors are presented. Table 1 compares the optical properties between (N-V) and two representative red and far-red fluorescent proteins. The in vivo structure of the latter protein is in form of tetramer and, therefore, has a size close to 10 nm. A simple calculation based on the molar extinction coefficient and the fluorescence quantum yield indicates that the brightness of a single (N-V) center is 2-fold as high as that of this far-red fluorescent protein, owing to the superb quantum yield of the atom-like fluorophore. Moreover, the (N-V) center fluoresces in the farther red region, where both autofluorescence and light scattering from the active biological environment are reduced to a greater extent. While compared to the infrared dye (such as IRDye-800CW in Table 1), the (N-V) center is a factor of 5 lower in brightness, this deficit can be readily compensated if each FND particle contains multiple (N-V) centers. It is the aim of this work to produce FNDs with a size comparable to those of far-red fluorescent proteins and concurrently containing as many (N-V) centers as possible. A previous experiment has shown that FNDs can be mass-produced with 40-keV Heþ irradiation of synthetic type Ib diamond nanocrystallites, followed by thermal annealing at 800 8C. For diamond powders with amedian size of 35 nm from a commercial source (MSY; Microdiamant), they usually have a broad size distribution and 10% of the particles are smaller than 15 nm. By optimizing the experimental parameters in differential centrifugation, we extracted sub-20-nm FNDs from the 35-nm ensembles after theHeþ irradiation andmulti-step centrifugation procedures. The sizes of these particles were finally characterized with dynamic light scattering (DLS), transmission electron microscopy (TEM), and atomic force microscopy (AFM). As revealed by DLS (Fig. 1a), the average diameter of the smallest FNDs that we have been able to extract with use of a standard centrifuge (Model 3700; Kubota) is 11.5 nm, accompanied with a narrow size deviation from 10 to 14 nm. TEM and AFM (Figs. S1 and S2 in Supporting Information) also confirmed independently the size distribution centered in the range of 10 nm at the single particle level. To make a direct comparison of the optical properties between the 11-nm FNDs and monomeric red fluorescent proteins (recombinant DsRed; BioVision), we characterized both specimens in solution with fluorescence correlation spectroscopy (FCS).

Recent advances in understanding the structures of medium-sized protonated water clusters

Understanding the structures of medium-sized protonated water clusters [H+(H2O) n ] has made a significant advancement recently thanks to the development of new experimental techniques and high-level computational methods. A combination of vibrational predissociation spectroscopy and ab initio calculations was shown to be effective in elucidating the structures of the clusters as a function of their temperature and size. The combined study revealed several intriguing features (such as symmetric hydrogen bonds) that could not be found for neutral water clusters. However, similar to its neutral counterpart, the number of stable isomers increases exponentially with cluster size (n), making direct structural identification of medium-sized clusters difficult. Despite the difficulties, both experimental and computational results indicated a smooth change in hydrogen-bond topology from tree-like, single-ring, multiple-ring to polyhedron-like structures (and their mixtures) as n increases from 5 to 28. The excess proton can be symmetrically hydrated at n = 6–8. Five-membered ring isomers can form at n = 7 and 8 as the low-lying minima. Only a single feature (∼3695 cm−1) in the free OH stretching region was observed for H+(H2O)21 and H+(H2O)28, suggesting that all surface water molecules are linked in a similar 3-coordinated (double-acceptor-single-donor, (AAD)) configuration in both “magic number” clusters. The clathrate-like structures open up at higher temperatures, as evidenced by the increased intensity of the free-OH stretching absorption band (∼3715 cm−1) of 2-coordinated (single-acceptor-single-donor, (AD)) water molecules. Further understanding of the structures and thermal properties of these clusters is gained through the studies with Monte Carlo (MC) and molecular dynamics simulations, ion reactivity and thermal dissociation measurements, as well as Ar tagging experiments. Contents PAGE 1. Introduction 554 2. Experiments 555 2.1. Hydrogen-bond topologies 556 2.2. “Magic number” clusters 559 2.3. Symmetric proton hydration 562 2.4. Cluster temperatures 564 3. Theories 567 3.1. Global minimum structures 567 3.2. Thermal and dynamical effects 572 4. Conclusion 574 Acknowledgements 451 References 451

Epitaxial growth of CO on NaCl(100) studied by infrared spectroscopy

Vibrational spectra of CO physisorbed onto well defined NaCl(100) surfaces were studied using a Fourier transform infrared interferometer. Structures of CO starting from the monolayer to multilayers were explored. At 31.5 K and a CO pressure of 1×10−6 mbar only the monolayer is formed. Polarization measurements confirm our earlier study that the monolayer CO molecules are aligned perpendicular to the NaCl(100) surface. Increasing the CO pressure to 7×10−6 mbar produces multilayer adsorption. The multilayer spectra closely resemble that of α‐CO absorption previously reported. The near perfect match of crystal structures and lattice constants of α‐CO and NaCl is reasoned to force the epitaxial growth of single crystal multilayers in our experiments. At 22 K the monolayer absorption is at 2155.01 cm−1 with a bandwidth (FWHH) of 0.26 cm−1. The two prominent features in the multilayer spectra at 22 K are assigned to the longitudinal optical (LO) mode at 2142.54 cm−1 and the transverse optical (TO) mode at 2138...

Infrared spectra and isomeric structures of hydroxide ion-water clusters OH- (H2O)1-5: a comparison with H3O (H2O)1-5

Size-selected hydroxide ion water tetramers and pentamers [OH-(H2O)4,5], produced by a supersonic expansion, have been investigated using vibrational predissociation spectroscopy in conjunction with ab initio calculations based on density functional theory (DFT). The observed spectra in the frequency range 2650–3850 cm−1 show some broad absorption bands attributed to the free and hydrogen bonded OH stretches of OH-(H2O)4,5 at an estimated cluster temperature of 170 K. DFT calculations performed at the B3LYP/6-31 + G* level reveal five and eight possible low lying isomeric forms for OH-(H2O)4 and OH-(H2O)5, respectively. The global minimum isomer of the tetramer is tri-solvated cyclic, which is energetically more stable than the tetra-solvated wheel-shaped form with an OH- ion at its centre. Compact cage-like lowest energy structures are found for the pentamer, in which the water molecules can act either as a single-donor-single-acceptor, as a double-proton-donor, or as a double-donor-single-acceptor in both the firs1183t a1192nd the second solvation shell of the OH- ion core. Interconversion among the isomers appears to be rapid as manifested in the observed spectra dominated by broad and congested absorptions. To understand the nature of spectral broadening and congestion, systemic comparisons of the results are made against those of the corresponding protonated cations, H+(H2O)n|1 and the corresponding halide anions, X-(H2O)n X = F, Cl, Br, and I. It is suggested that the spectral complexities observed for OH-(H2O)4,5 are predominantly a result of sampling configurations with a large distribution of Osolvent-Oion-Osolvent angles and Osolvent … H-Osolvent distances between water molecules in the firs1175t a1181nd/or second hydration shells, together with the existence of more than one isomer in the supersonic expanson and rapid isomeric interconversion among them.