Hongyun Wang

Resolving Sub-Diffraction Limit Encounters in Nanoparticle Tracking Using Live Cell Plasmon Coupling Microscopy

We use plasmon coupling between individual gold nanoparticle labels to monitor subdiffraction limit distances in live cell nanoparticle tracking experiments. While the resolving power of our optical microscope is limited to approximately 500 nm, we improve this by more than an order of magnitude by detecting plasmon coupling between individual gold nanoparticle labels using a ratiometric detection scheme. We apply this plasmon coupling microscopy to resolve the interparticle separations during individual encounters of gold nanoparticle labeled fibronectin-integrin complexes in living HeLa cells.

Scavenger Receptor Mediated Endocytosis of Silver Nanoparticles into J774A.1 Macrophages is Heterogeneous

We investigated the scavenger receptor mediated uptake and subsequent intracellular spatial distribution and clustering of 57.7 ± 6.9 nm diameter silver nanoparticles (zeta-potential = -28.4 mV) in the murine macrophage cell line J774A.1 through colorimetric imaging. The NPs exhibited an overall red-shift of the plasmon resonance wavelength in the cell ensemble as function of time and concentration, indicative of intracellular NP agglomeration. A detailed analysis of the NP clustering in individual cells revealed a strong phenotypic variability in the intracellular NP organization on the single cell level. Throughout the observation time of 24h cells containing non- or low-agglomerated NPs with a characteristic blue color coexisted with cells containing NPs with varying degrees of agglomeration, as evinced by distinct spectral shifts of their resonance wavelengths. Pharmacological inhibition studies indicated that the observed differences in intracellular NP organization resulted from coexisting actin- and clathrin-dependent endocytosis mechanisms in the macrophage population. Correlation of intracellular NP clustering with macrophage maturity marker (F4/80, CD14) expression revealed that differentiated J774A.1 cells preferentially contained compact NP agglomerates, whereas monocyte-like macrophages contained non-agglomerated NPs.

Illuminating epidermal growth factor receptor densities on filopodia through plasmon coupling.

Filopodia have been hypothesized to act as remote sensors of the cell environment, but many details of the sensor function remain unclear. We investigated the distribution of the epidermal growth factor (EGF) receptor (EGFR) density on filopodia and on the dorsal cell membrane of A431 human epidermoid carcinoma cells using a nanoplasmonic enabled imaging tool. We targeted cell surface EGFR with 40 nm diameter Au nanoparticles (NPs) using a high affinity multivalent labeling strategy and determined relative NP binding affinities spatially resolved through plasmon coupling. Distance-dependent near-field interactions between the labels generated a NP density (ρ)-dependent spectral response that facilitated a spatial mapping of the EGFR density distribution on subcellular length scales in an optical microscope in solution. The measured ρ values were significantly higher on filopodia than on the cellular surface, which is indicative of an enrichment of EGFR on filopodia. A detailed characterization of the spatial distribution of the NP immunolabels through scanning electron microscopy (SEM) confirmed the findings of the all-optical plasmon coupling studies and provided additional structural details. The NPs exhibited a preferential association with the sides of the filopodia. We calibrated the ρ-dependent spectral response of the Au immunolabels through correlation of optical spectroscopy and SEM. The experimental dependence of the measured plasmon resonance wavelength (λ(res)) of the interacting immunolabels on ρ was well described by the fit λ(res) = 595.0 nm - 46.36 nm exp(-ρ/51.48) for ρ ≤ 476 NPs/μm(2). The performed correlated spectroscopic/SEM studies pave the way toward quantitative immunolabeling studies of EGFR and other important cell surface receptors in an optical microscope.

Optical Sizing of Immunolabel Clusters through Multispectral Plasmon Coupling Microscopy

The wavelength dependent scattering cross sections of self-assembled silver nanoparticle clusters of known size (n) were measured on five different wavelength channels between 427 and 510 nm through correlation of multispectral imaging and scanning electron microscopy. A multivariate statistical analysis of the spectral response of this training set provided a correlation between spectral response and cluster size and enabled a classification of new measurements into four distinct nanoparticle association levels (I1-I4) whose compositions were dominated by monomers (I1), dimers (I2), trimers and tetramers (I3), and larger clusters (I4), respectively. One potential application of the optical sizing approach is to map association levels of silver immunolabels on cellular surfaces. We demonstrate the feasibility of this approach using silver immunolabels targeted at the epidermal growth factor receptor on A431 cells in a proof of principle experiment. The ability to measure immunolabel association levels on subcellular length scales in an optical microscope provides new opportunities for experimentally assessing receptor density distributions on living cells in solution.

Monitoring Cellular Trafficking of Nanoparticle Cargo in Murine Macrophages Through Plasmon Coupling Microscopy

A detailed analysis of silver nanoparticle (NP) uptake and trafficking in the murine macrophage cell line J774A.1 through spectral analysis of the resonance wavelength of the metal NP cargo is presented. The NP spectra reveal a strong phenotypic variability in the NP uptake and processing on the single cell level. Cells containing non- or low-agglomerated NPs are found to coexist with cells containing NPs of varying degrees of NP agglomeration, clearly indicated by a spectral red-shift in the resonance wavelength. Pharmacological inhibition studies indicate that the observed differences in the intracellular NP organization result from coexisting actin- and clathrin-dependent endocytosis mechanisms. Correlation with fluorescence macrophage maturity markers shows that differentiated J774A.1 macrophages preferentially contain compact NP agglomerates, whereas monocyte-like macrophages contain non-agglomerated NPs.Copyright

Silver plasmon rulers as probes in polarization-resolved plasmon coupling microscopy

The distance dependent interactions between individual noble metal nanoparticles enable active plasmonic nanostructures with applications in microscopy, sensing and imaging. If two nanoparticles approach each other close enough for their plasmons to couple, the resonance wavelength of the dimer red-shifts. This effect is utilized in plasmon coupling microscopy (PCM) to resolve subdiffraction limit contacts. We apply polarization resolved PCM to monitor changes in the orientation and interparticle separation of individual silver plasmon rulers during their lateral diffusion on a plasma membrane. The ability to track the position of individual silver plasmon rulers and to simultaneously monitor their rotation and separation in PCM makes plasmon ruler probes for the local structure of the supporting membrane.

Mapping the Spatial Distribution of Cell Surface Receptors with Plasmon Coupling Microscopy

A multispectral imaging modality is described for a quantitative analysis of plasmon coupling between noble metal immunolabels. The approach provides information about the spatial organization of the labels and thus of targeted receptor densities.