Kort Travis

Plasmon resonance coupling of metal nanoparticles for molecular imaging of carcinogenesis in vivo

An effective cancer control strategy requires improved early detection methods, patient-specific drug selection, and the ability to assess response to targeted therapeutics. Recently, plasmon resonance coupling between closely spaced metal nanoparticles has been used to develop ultrasensitive bioanalytical assays in vitro. We demonstrate the first in vivo application of plasmon coupling for molecular imaging of carcinogenesis. We describe molecular-specific gold bioconjugates to image epidermal growth factor receptor (EGFR); these conjugates can be delivered topically and imaged noninvasively in real time. We show that labeling with gold bioconjugates gives information on the overexpression and nanoscale spatial relationship of EGF receptors in cell membranes, both of which are altered in neoplasia. EGFR-mediated aggregation of gold nanoparticles in neoplastic cells results in more than a 100-nm color shift and a contrast ratio of more than tenfold in images of normal and precancerous epithelium in vivo, dramatically increasing contrast beyond values reported previously for antibody-targeted fluorescent dyes.

Dynamic Imaging of Molecular Assemblies in Live Cells Based on Nanoparticle Plasmon Resonance Coupling

We used molecular-specific gold nanoparticles to monitor epidermal growth factor receptors (EGFR) in live A431 cells over time. Dark-field hyperspectral imaging, electron microscopy, and electrodynamic modeling were used to correlate optical properties of EGFR-bound plasmonic nanoparticles with receptor regulation state. We showed that receptor trafficking resulted in a progressive red shift of greater than 100 nm in the nanoparticle plasmon resonance wavelength over a time period of 60 min. Furthermore, we demonstrated that changes in peak scattering wavelengths of gold nanoparticles from 546 +/- 15 to 574 +/- 20, and to 597 +/- 44 nm are associated with EGFR trafficking from the cell membrane, to early endosomes, and to late endosomes/multivesicular bodies, respectively. Finally, we used the changes in scattering spectra of EGFR-bound nanoparticles and a straightforward statistical analysis of RGB-channel color images of labeled cells to create near real-time maps of EGFR regulatory states in living cells.

Controlled Assembly of Biodegradable Plasmonic Nanoclusters for Near-Infrared Imaging and Therapeutic Applications

Metal nanoparticles with surface plasmon resonance (SPR) in the near-infrared region (NIR) are of great interest for imaging and therapy. Presently, gold nanoparticles with NIR absorbance are typically larger than 50 nm, above the threshold size of approximately 5 nm required for efficient renal clearance. As these nanoparticles are not biodegradable, concerns about long-term toxicity have restricted their translation into the clinic. Here, we address this problem by developing a flexible platform for the kinetically controlled assembly of sub-5 nm ligand-coated gold particles to produce metal/polymer biodegradable nanoclusters smaller than 100 nm with strong NIR absorbance for multimodal application. A key novel feature of the proposed synthesis is the use of weakly adsorbing biodegradable polymers that allows tight control of nanocluster size and, in addition, results in nanoclusters with unprecedented metal loadings and thus optical functionality. Over time, the biodegradable polymer stabilizer degrades under physiological conditions that leads to disassembly of the nanoclusters into sub-5 nm primary gold particles which are favorable for efficient body clearance. This synthesis of polymer/inorganic nanoclusters combines the imaging contrast and therapeutic capabilities afforded by the NIR-active nanoparticle assembly with the biodegradability of a polymer stabilizer.

Polarization microscopy with stellated gold nanoparticles for robust, in-situ monitoring of biomolecules

Advances in plasmonic nanoparticle synthesis afford new opportunities for biosensing applications. Here, we apply a combination of a new type of plasmonic nanomaterial – stellated nanoparticles, and polarization-sensitive darkfield microscopy for detecting molecular assemblies and tracking of individual epidermal growth factor receptors within single live cells with high signal-to-background ratio. Depolarization of linear polarized light by stellated nanoparticles is over 15-fold more efficient than similarly-sized spheroidal nanoparticles. This efficient light depolarization allows robust detection of molecules labeled with stellated nanoparticles in cross-polarized imaging where the intrinsic light scattering from cells is significantly reduced. The imaging can be carried out with single molecule sensitivity for essentially unlimited time with no signal degradation.

Polaro–cryptic mirror of the lookdown as a biological model for open ocean camouflage

With no object to hide behind in 3D space, the open ocean represents a challenging environment for camouflage. Conventional strategies for reflective crypsis (e.g., standard mirror) are effective against axially symmetric radiance fields associated with high solar altitudes, yet ineffective against asymmetric polarized radiance fields associated with low solar inclinations. Here we identify a biological model for polaro–crypsis. We measured the surface-reflectance Mueller matrix of live open ocean fish (lookdown, Selene vomer) and seagrass-dwelling fish (pinfish, Lagodon rhomboides) using polarization-imaging and modeling polarization camouflage for the open ocean. Lookdowns occupy the minimization basin of our polarization-contrast space, while pinfish and standard mirror measurements exhibit higher contrast values than optimal. The lookdown reflective strategy achieves significant gains in polaro–crypsis (up to 80%) in comparison with nonpolarization sensitive strategies, such as a vertical mirror. Lookdowns achieve polaro–crypsis across solar altitudes by varying reflective properties (described by 16 Mueller matrix elements mij) with incident illumination. Lookdowns preserve reflected polarization aligned with principle axes (dorsal–ventral and anterior–posterior, m22 = 0.64), while randomizing incident polarization 45° from principle axes (m33 = –0.05). These reflectance properties allow lookdowns to reflect the uniform degree and angle of polarization associated with high-noon conditions due to alignment of the principle axes and the sun, and reflect a more complex polarization pattern at asymmetrical light fields associated with lower solar elevations. Our results suggest that polaro–cryptic strategies vary by habitat, and require context-specific depolarization and angle alteration for effective concealment in the complex open ocean environment.

Open-ocean fish reveal an omnidirectional solution to camouflage in polarized environments.

Disappearing act Unlike coastal regions and reefs, the open ocean is mostly empty. Many fish species, nonetheless, spend most of their lives there. Such emptiness makes camouflage exceedingly difficult, so how does an organism hide in water filled with bouncing and reflected light? Brady et al. show that some families of fish have evolved skin that reflects and polarizes light, allowing them to blend into their mirrorlike conditions more easily. These results help to explain the silvery coloration found in sea-living fish across the worlds oceans. Science, this issue p. 965 Light-reflecting and -polarizing platelets in their skin permit fish to blend into the mirrorlike open ocean. Despite appearing featureless to our eyes, the open ocean is a highly variable environment for polarization-sensitive viewers. Dynamic visual backgrounds coupled with predator encounters from all possible directions make this habitat one of the most challenging for camouflage. We tested open-ocean crypsis in nature by collecting more than 1500 videopolarimetry measurements from live fish from distinct habitats under a variety of viewing conditions. Open-ocean fish species exhibited camouflage that was superior to that of both nearshore fish and mirrorlike surfaces, with significantly higher crypsis at angles associated with predator detection and pursuit. Histological measurements revealed that specific arrangements of reflective guanine platelets in the fish’s skin produce angle-dependent polarization modifications for polarocrypsis in the open ocean, suggesting a mechanism for natural selection to shape reflectance properties in this complex environment.

Cancer imaging and therapy with metal nanoparticles

Nanotechnology offers unique opportunities for cancer detection, therapy and the ability to monitor therapeutic interventions. This potential has to be analyzed in context of challenges that need to be overcome in translation of nanoparticles to clinical applications including specific delivery in tissues and clearance from the body. Here, we will present a case study of plasmonic nanoparticles in cancer imaging and therapy.

Phenomenology of optical scattering from plasmonic aggregates for application to biological imaging and clinical therapeutics

Near-field coupling between plasmonic resonant nanoparticles and the associated shifts in scattering spectra enables the accomplishment of unprecedented observation of the co-localization dynamics of in-situ biomolecules on nanometer length-scales. We have recently shown that resonant nanoparticles conjugated to antibodies for cell-surface receptors provide a sensitive probe allowing the unambiguous resolution of not only the time sequence, but also the details of the intracellular pathway, for receptor-mediated endocytosis in live cells. In terms of general principles, the classical electrodynamics determining the scattering cross-section for nanoparticle aggregates is straightforward. However, the specifics of the angular dependence of the differential cross-section at a single wavelength, the wavelength dependence of this cross-section, and the correct implementation and interpretation of statistical averages of cross-section properties over an ensemble of aggregate morphologies are generally quite complicated, and in fact are often misinterpreted in the literature. Despite this complexity, we have constructed a set of few-parameter formulae describing optical scattering from nanoparticle aggregates by judicious combination of experimental results with extensive, near-exact simulation using the T-matrix technique. These phenomenological results facilitate the practical use of nanoparticle aggregates for biological measurement and clinical therapeutic applications.

Model-based design of optical diagnostic instrumentation

Biophotonics methods are attractive since they allow for the non-invasive diagnosis of cancer. Experiments were carried out to investigate the feasibility of detecting early pre-cancer using optical spectroscopy. However, optimization of instrumentation design parameters remains challenging because of the lack of metrics to evaluate the performance of certain design parameters. For example, although using angled-collection geometry has been shown to collect depth sensitive spatial origins, the performance of devices with angled-collection geometries are not well characterized or quantified. In this study, we use a polarization-sensitive Monte Carlo simulation (Pol-MC) to aid in the design of instrumentation for the early detection of epithelial cancer. The tissue is modeled in layers: (0) air outside the tissue, (1) epithelial layer, (2) thin pre-cancer layer of cells, (3) thin basement membrane, implemented as a thin transparent layer, and (4) the stroma, implemented as a thick layer of scattering material. We propose a new metric, Target Signal Ratio (TSR), to evaluate the proportion of signal that is scattered from a target layer, which is the basal/pre-cancer layer. This study is a proof-of-concept for the application of computational techniques to facilitate instrument design.

Biodegradable near-infrared plasmonic nanoclusters for biomedical applications

Nanoparticles such as gold and silver with plasmonic resonances in the near-infrared (NIR) optical region, where soft tissue is the most transparent, are of great interest in biomedical applications. A major roadblock in translation of inorganic nanoparticles to clinical practice for systemic targeting of disease is their nonbiodegradable nature. In addition, gold nanoparticles that absorb in the NIR are typically greater than 50 nm, which is above the threshold size of 5.5 nm required for effective excretion from the body. Here we describe a new class of biodegradable gold nanoparticles with plasmon resonances in the NIR region. The synthesis is based on controlled assembly of very small (less than 5 nm) primary gold particles into nanoclusters with sub-100 nm overall diameter and an intense NIR absorbance. The assembly is mediated by biodegradable polymers, polyethylene glycol (PEG) and polylactic acid (PLA) copolymer, and small capping ligands on the constituent nanoparticles. Nanoclusters deaggregate into sub-5nm primary gold particles upon biodegradation of the polymer binder in live cells over one week, as shown by dark-field reflectance and hyperspectral imaging.