Jeeseong Hwang

Domains in Cell Plasma Membranes Investigated by Near-Field Scanning Optical Microscopy

Near-field scanning optical microscopy (NSOM) uses the near-field interaction of light from a sharp fiber-optic probe with a sample of interest to image surfaces at a resolution beyond the diffraction limit of conventional optics. We used NSOM to image fluorescently labeled plasma membranes of fixed human skin fibroblasts, either dried or in buffer. A patchy distribution of a fluorescent lipid analog suggestive of lipid domains was observed in the fixed, dried cells. The sizes of these patches were consistent with the sizes of domains implied by fluorescence photobleaching recovery measurements. Patches of fluorescent lipid analog were not spatially correlated with patches of transmembrane proteins, HLA class I molecules labeled with fluorescent antibody; the patchiness of the HLA class I molecules was on a smaller scale and was not localized to the same regions of membrane as the lipid analog. Sizes of HLA patches were deduced from a two-dimensional spatial autocorrelation analysis of NSOM images that resolved patches with radii of approximately 70 and approximately 600 nm on fixed, dried cells labeled with IgG and 300-600 nm on cells labeled with Fab and imaged in buffer. The large-size patches were also resolved by far-field microscopy. Both the spatial autocorrelation analysis and estimates from fluorescence intensity indicate that the small patches seen on fixed, dried cells contain approximately 25-125 HLA-I molecules each.

Nanoscale complexity of phospholipid monolayers investigated by near-field scanning optical microscopy

Near-field scanning optical microscopy of phospholipid monolayers doped with fluorescent lipid analogs reveals previously undescribed features in various phases, including a concentration gradient at the liquid-expanded/liquid-condensed domain boundary and weblike structures in the solid-condensed phase. Presumably, the web structures are grain boundaries between crystalline solid lipid. These structures are strongly modulated by the addition of low concentrations of cholesterol and ganglioside GM1 in the monolayer.

Band 3 Modifications in Plasmodium Falciparum-Infected AA and CC Erythrocytes Assayed by Autocorrelation Analysis Using Quantum Dots

The molecular stability of hemoglobin is critical for normal erythrocyte functions, including oxygen transport. Hemoglobin C (HbC) is a mutant hemoglobin that has increased oxidative susceptibility due to an amino acid substitution (β6: Glu to Lys). The growth of Plasmodium falciparum is abnormal in homozygous CC erythrocytes in vitro, and CC individuals show innate protection against severe P. falciparum malaria. We investigated one possible mechanism of innate protection using a quantum dot technique to compare the distribution of host membrane band 3 molecules in genotypically normal (AA) to CC erythrocytes. The high photostability of quantum dots facilitated the construction of 3D cell images and the quantification of fluorescent signal intensity. Power spectra and 1D autocorrelation analyses showed band 3 clusters on the surface of infected AA and CC erythrocytes. These clusters became larger as the parasites matured and were more abundant in CC erythrocytes. Further, average cluster size (500 nm) in uninfected (native) CC erythrocytes was comparable with that of parasitized AA erythrocytes but was significantly larger (1 μm) in parasitized CC erythrocytes. Increased band 3 clustering may enhance recognition sites for autoantibodies, which could contribute to the protective effect of hemoglobin C against malaria.

Low-cost, high-throughput, automated counting of bacterial colonies.

Research involving bacterial pathogens often requires enumeration of bacteria colonies. Here, we present a low‐cost, high‐throughput colony counting system consisting of colony counting software and a consumer‐grade digital camera or document scanner. We demonstrate that this software, called “NICE” (NISTs Integrated Colony Enumerator), can count bacterial colonies as part of a high‐throughput multiplexed opsonophagocytic killing assay used to characterize pneumococcal vaccine efficacy. The results obtained with NICE correlate well with the results obtained from manual counting, with a mean difference of less than 3%. NICE is also rapid; it can count colonies from multiple reaction wells within minutes and export the results to a spreadsheet for data processing. As this program is freely available from NIST, NICE should be helpful in bacteria colony enumeration required in many microbiological studies, and in standardizing colony counting methods. Published 2010 Wiley‐Liss, Inc.

Quantitative Characterization of Quantum Dot-Labeled Lambda Phage for Escherichia coli Detection

We characterize CdSe/ZnS quantum dot (QD) binding to genetically modified bacteriophage as a model for bacterial detection. Interactions among QDs, lambda (λ) phage, and Escherichia coli are examined by several cross‐validated methods. Flow and image‐based cytometry clarify fluorescent labeling of bacteria, with image‐based cytometry additionally reporting the number of decorated phage bound to cells. Transmission electron microscopy, image‐based cytometry, and electrospray differential mobility analysis allow quantization of QDs attached to each phage (4–17 QDs) and show that λ phage used in this study exhibits enhanced QD binding to the capsid by nearly a factor of four compared to bacteriophage T7. Additionally, the characterization methodology presented can be applied to the quantitative characterization of other fluorescent nanocrystal‐biological conjugates. Biotechnol. Bioeng. 2009;104: 1059–1067. Published 2009 Wiley Periodicals, Inc.

Design and optimization of a near-field scanning optical microscope for imaging biological samples in liquid

We describe a near-field scanning optical microscope capable of imaging biological samples in liquid. The microscope uses a straight optical fiber near-field probe and optical shear-force feedback for tip-sample distance regulation. Physical aspects of the design are discussed, and phenomena related to operation in liquid are revealed. Careful calibration of the instrument in air and in liquid is shown, and for the first time to our knowledge, near-field fluorescence images of a biological cell in liquid are presented.

Atomically flat gold films grown on hot glass

The results of a scanning tunneling microscope (STM) study of the morphology of Au films thermally evaporated onto heated glass substrates are presented. Au films of thickness 20–80 nm were evaporated onto Corning glass cover slips at temperatures of 20–465 °C. Before the evaporation, the glass substrates were prebaked at 300–400 °C for 12 h to remove surface contamination. Grain size and surface roughness of the films have been measured, and the best results were obtained with Au films 80 nm thick evaporated onto 300 °C substrates. These films have 250‐nm‐diam grains with large, atomically flat tops exhibiting step‐free terraces as large as 200×200 nm2. These results are comparable to the best reported results for Au grown on heated mica and show that epitaxial growth is not required for the production of films with large atomically flat regions. This study of Au on glass was motivated by failed attempts to produce flat films of gold on mica using recipes in the literature. Possible reasons for the poor ...

Absorption-Based Hyperspectral Imaging and Analysis of Single Erythrocytes

We report an absorption-based hyperspectral imaging and analysis technique to resolve unique physicochemical characteristics of subcellular substances in single erythrocytes. We constructed a microscope system installed with a spectral light engine capable of controlling the spectral shape of the illumination light by a digital micromirror device. The hyperspectral imaging system and the sequential maximum angle convex cone algorithm allow us to extract unique spectral signatures (i.e., endmembers) for different types of hemoglobin, such as oxyhemoglobin, methemoglobin, and hemozoin, and scatter from cell membrane in single erythrocytes. Further statistical endmember analysis, conducted on the hyperspectral image data, provides the abundances of specific endmembers, which can be used to build intracellular maps of the distribution of substances of interest. In addition, we perform modeling based on Mie theory to explain the scattering signatures as a function of scattering angle. The developed imaging and analysis technique enables label-free molecular imaging of endogenous biomarkers in single erythrocytes in order to build oxymetric standards on a cellular level and ultimately for in vivo as well.

Water-soluble DNA-wrapped single-walled carbon-nanotube/quantum-dot complexes.

In recent years, carbon nanotubes (CNTs), especially singlewalled carbon nanotubes (SWCNTs), have attracted much attention due to their unique properties and potential towards broad real-world applications. The integration of SWCNTs with other unique nanoscale luminescent materials, such as quantum dots (QDs), has enabled the manufacture of many novel nanocomposite materials with enhanced structural, mechanical, optical, and chemical properties. The performance of these composite materials strongly depends upon the properties of the individual components and additives as well as the conjugation chemistry required to assemble them into composite hybrids. Therefore, a variety of new techniques have been developed to modify the optical, mechanical, chemical, and electrical properties of SWCNTs to control the properties of the final composite materials. Among the additives to SWCNT-based composites, novel nanoparticles (NPs) have been increasingly employed. Functionalized NPs can be designed to covalently bind to the functional groups expressed on the sidewalls or ends of

Developing digital tissue phantoms for hyperspectral imaging of ischemic wounds

Hyperspectral imaging has the potential to achieve high spatial resolution and high functional sensitivity for non-invasive assessment of tissue oxygenation. However, clinical acceptance of hyperspectral imaging in ischemic wound assessment is hampered by its poor reproducibility, low accuracy, and misinterpreted biology. These limitations are partially caused by the lack of a traceable calibration standard. We proposed a digital tissue phantom (DTP) platform for quantitative calibration and performance evaluation of spectral wound imaging devices. The technical feasibility of such a DTP platform was demonstrated by both in vitro and in vivo experiments. The in vitro DTPs were developed based on a liquid blood phantom model. The in vivo DTPs were developed based on a porcine ischemic skin flap model. The DTPs were projected by a Hyperspectral Image Projector (HIP) with high fidelity. A wide-gap 2nd derivative oxygenation algorithm was developed to reconstruct tissue functional parameters from hyperspectral measurements. In this study, we have demonstrated not only the technical feasibility of using DTPs for quantitative calibration, evaluation, and optimization of spectral imaging devices but also its potential for ischemic wound assessment in clinical practice.