This PDF file contains the front matter associated with SPIE Proceedings Volume 6565, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

We report the development of a novel methodology for patterning of nanostructured sensory materials using multi-dimensional microstructured support platforms for optical bioimaging applications. Specifically, the support platforms are fabricated using direct-write technique and sol-gel derived xerogel thin-films to form the sensor materials. This creates a simple and versatile method for developing complex 3-D microstructures that have the combined capabilities of biochemical sensing, microfluidic sample distribution for sensor arrays, and direct integration with Complimentary Metal-Oxide Semiconductor (CMOS) Integrated Circuits (ICs) used for sensor signal detection and processing. More importantly, this methodology would enable the development of large-scale arrayed sensing platforms for applications in cell-culture analysis and tissue imaging. The configuration and fabrication of the proposed microstructures, which consist of planar ridge and hollow waveguides, will be described in detail. As a prototype implementation, we demonstrate direct-write ridge waveguide support structures coated with luminophore-doped xerogels that are responsive to gaseous oxygen (O₂) concentration.

Biomolecular motors, such as the motor protein kinesin, are simultaneously objects of scientific inquiry and components for nanotechnology. The investigation of the properties of a biomolecular motor is challenging, since it is a dynamic nanoscale object but at the same time soft and fragile. Photonic techniques are well suited to these investigations due to their compatibility with an aqueous environment and their non-destructive character, however their resolution is often insufficient. We adapted Fluorescence Interference Contrast (FLIC) microscopy to the imaging of microtubules transported by kinesin motors (PNAS vol. 103, p. 15812) and achieved nm-resolution in the z-direction. This advance provided insights into the role of the kinesin tail for the functioning of the motor in vivo, but also enabled us to determine the "ground clearance" of molecular shuttles powered by kinesin motors. Kinesin-driven molecular shuttles, in turn, enable the design of highly integrated bionanodevices. Photons are the most suitable tool to communicate with such devices, since they can address molecules and nanoparticles packaged into the devices without the need for a physical connection.

Motility assays are the tools of choice for the studies regarding the motility of protein molecular motors in vitro. Despite their wide usage, some simple, but fundamental issues still need to be specifically addressed in order to achieve the best and the most meaningful motility analyses. Several tracking methods used for the study of motility have been compared. By running different statistical analyses, the impact of space versus time resolution was also studied. It has been found that for a space resolution of 80 nm and 145 nm per pixel for kinesin-microtubule and actomyosin assays, respectively, the best time resolution was ~0.9 and ~10 frame per second, respectively. A rough relationship - Ratio_A and Ratio_M - between space and time resolutions and velocity for actin filaments and microtubules, respectively, was found. The motility parameters such as velocity, acceleration and deflection angle were statistically analysed in frequency distribution and time domain graphs for both motors assays. One of the aims of these analyses was to study if one or two populations were present in either assay. Particularly for actomyosin assays, electric fields varying from 0 to ~10000 Vm^-1 were applied and the previous parameters and the angle between filaments motion and the electric field vector were also statistically analysed. It was observed that this angle was reduced by ~55º with ~5900 Vm^-1. The overall behaviour of the motors was discussed bearing in mind both present and previous results and some physio-biological characteristics. Kinesin-microtubule and actomyosin (simple and electric fields) assays were compared. Some new experiments are suggested in order to accomplish a better understanding of these motors and optimise their role in the applications that depend on them.

We introduce a new aperture-type near-field scanning optical microscopy (NSOM) system, which rely on large area (e.g., > 200 x 200 nm) aperture geometries that have sharp corners. The spatial resolution of this new near-field imaging modality is not determined by the size of the aperture, but rather by the sharpness of the corners of the large aperture. This approach significantly improves the light throughput of the near-field probe and therefore increases the optical signal-to-noise ratio (SNR). Here we discuss the basics of this new near-field microscopy approach and illustrate both theoretically and experimentally that an array of detectors can be utilized to further improve the SNR of the near-field image.

Molecular Interferometric Imaging (MI2) is a sensitive detection platform for direct optical detection of immobilized biomolecules. It is based on inline common-path interferometry combined with far-field optical imaging. The substrate is a simple thermal oxide on a silicon surface with a thickness at or near the quadrature condition that produces a π/2 phase shift between the normal-incident wave reflected from the top oxide surface and the bottom silicon surface. The presence of immobilized or bound biomolecules on the surface produces a relative phase shift that is converted to a far-field intensity shift and is imaged by a reflective microscope onto a CCD camera. Shearing interferometry is used to remove the spatial 1/f noise from the illumination to achieve shot-noise-limited detection of surface dipole density profiles. The lateral resolution of this technique is diffraction limited at 0.4 micron, and the best longitudinal resolution is 10 picometers. The minimum detectable mass at the metrology limit is 2 attogram, which is 8 antibody molecules of size 150 kDa. The corresponding scaling mass sensitivity is 5 fg/mm compared with 1 pg/mm for typical SPR sensitivity. We have applied MI2 to immunoassay applications, and real-time binding kinetics has been measured for antibody-antigen reactions. The simplicity of the substrate and optical read-out make MI2 a promising analytical assay tool for high-throughput screening and diagnostics.

We employed atomic force microscopy (AFM) with bias control to fabricate oxided nanopatterns on silicon surface with feature size down to 50nm. The relationship of silicon dioxide nanopatterns against humidity was studied and then the optimal parameter was used to make oxide nanoarry for interaction of biotin and streptavidin. The scanning function of AFM was utilized to verify the different height of biomolecules. According to our experimental results, using nano biochip of silicon dioxide can decrease the monitoring scale to nanometer and can be the nano-platform for monitoring the behavior of biomolecular interaction. We anticipate mimicking the correlation of single molecular behavior and an array of biomolecular behavior to understand the coincidence of them.

A new method for using a non-selectively filled hollow-core photonic crystal fiber (HC-PCF) as a sensitive Raman spectroscopy platform suitable for biosensing applications is presented. A 1550 HC-PCF was completely filled with ethanol (core and cladding holes). Using a 785nm excitation laser, the Raman spectrum of ethanol in the fiber core was obtained and compared with the equivalent Raman spectrum of an ethanolfilled cuvette. Using a relatively short 9.5cm length of HC-PCF, a Raman signal enhancement factor of 40 over a bulk solution of ethanol was observed under the same excitation conditions. The small sample volume utilized and longer interaction length provides the potential for compact, sensitive, and low-power Raman sensing of biological materials

Fluorescence techniques rely on measurement of relative fluorescence units and require calibration to obtain reliable and comparable quantitative data. Fluorescent immunoassays are a very sensitive and convenient method of choice for rapid detection of biotoxins, such as ricin. Here we present the application of magnetic luminescent nanoparticles (MLNPs) with a magnetic core of Fe₃O₄ and a fluorescent shell of Eu:Gd₂O₃ as carriers for a nanobead-immunoassay for the detection of ricin with internal calibration. A sandwich immunoassay for ricin was performed on the surface of the MLNPs. The particles were functionalized with capture polyclonal antibodies. Anti-ricin antibodies labeled with Alexa Fluor dye were used as the detecting antibodies. After magnetic extraction, the amount of ricin bound to the particle surface was quantified and related to the fluorescence signal of the nanoparticles. In this new platform, the MLNPs have three main functions: (1) a probe for the specific extraction of the target analyte from the sample; (2) a carrier in the quantitative immunoassay with magnetic separation; and (3) an internal standard in the fluorescence measurement of the dye reporter. The MLNPs serve as an internal control for the total analysis including extraction and assay performance. This approach eliminates the experimental error inherent in particle extraction and measurement of absolute organic dye fluorescence intensities. All fluorescent measurements were performed in a microplate reader. The standard curve for ricin had a dynamic range from 20 ng/ml to 100 μg/ml with a detection limit of 5 ng/ml. The configuration that has been developed can be easily adapted to a high throughput miniaturized system.

We demonstrate preparations of zinc porphyrin nanoparticles by reprecipitation method and their spectroscopic analysis by dark-field light scattering microspectroscopy. The size distribution of the prepared nanoparticles was 80-150 nm. By using dark-field illumination the nanoparticles could be observed as bright points in dark background and could be examined by their Rayleigh scattering spectra at single particle level. The spectra differed from particle to particle, which would be ascribed to their size and crystalline phase difference. Thus we have performed this single particle spectroscopic technique to remove the ambiguity about the spectroscopic information owing to distributions of particles and to improve the space selectivility. In addition, we have successfully demonstrated the detection of amine molecules in water at single particle level. These results indicate that the detection technique using the single porphyrin nanoparticles can be applied to chemical and biological sensors with nanometer scale.

We have investigated the effect of application of gold nanoshells with a 150 nm silica core size and 25 nm thick gold coating on optical properties of skin. We have analyzed the possibility of using these particles as a contrasting agent for optical coherence tomography (OCT). A set of Monte Carlo calculations was performed in order to simulate the images of skin before and after application of the nanoshells for a skin model close to that in vivo. We investigated the mechanisms of boundary contrasting between tissue layers with different optical properties in the presence of gold nanoshells on two-layer agar gel phantom. Gold nanoshells were also applied on the skin surface in vivo. Gold-silica nanoshells caused an increase in the intensity of OCT signal, brightness of the superficial part of the dermis, contrast between dermis layers and contrast of hair follicles and glands in the OCT image. The contrasting effects of the gold nanoshells lasted up to 24 hours of observation.

We have used flow-cytometry together with computational modeling of quantum dot portioning during cell division to identify population distributions of proliferating cells. The objective has been to develop a robust assay of integrated cellular fluorescence which reports the extent of cellular bifurcation within a complex population and potentially provides profiles of drug resistance, cell clonality and levels of aneuploidy in tumour cells. The implementation of a data analysis program based on genetic algorithms provides a complete description of the proliferation dynamics and gives values for the inter-mitotic time, the partitioning ratio of quantum dots between daughter cells and their associated deviations.

Despite convincing evidence for hyperthermic radiosensitization, the invasive means of achieving and monitoring hyperthermia and the lack of good thermal dosimetry have hindered its use in routine clinical practice. A non-invasive method to generate and monitor hyperthermia would provide renewed enthusiasm for such treatments. Near-infrared absorbing gold nanoshells have been shown to accumulate preferentially in tumors via the enhanced permeability and retention effect and have been used for thermal ablation of tumors. We evaluated the use of these nanoshells to generate hyperthermia to evaluate the anti-tumor effects of combining gold nanoshell mediated hyperthermia with radiotherapy. Laser settings were optimized for hyperthermia in a mouse xenograft model to achieve a temperature rise of 40- 41°C in the tumor periphery and 37-38°C (ΔT=4-5°C) deeper within the tumors. The ΔT measurements were verified using both thermocouple and magnetic resonance thermal imaging (MRTI) temperature measurements. Tumor re-growth delay was estimated by measuring tumor size after treatment with radiation (10Gy single dose), hyperthermia (15 minutes at 40°C), and hyperthermia followed by radiation and control. Significant difference (p <0.05) in the tumor volume doubling time was observed between the radiation group (13 days) and combination treatment group (25 days). The immunofluorescence staining for the hypoxic, proliferating cells and the vasculature corroborated our hypothesis that the radiosensitization is in part mediated by increased initial perfusion and subsequent collapse of vasculature that leads to acute inflammatory response in the tumor. The increased vascular perfusion immediately after gold nanoshell mediated hyperthermia is confirmed by dynamic contrast enhanced magnetic resonance imaging.

Laser induced thermal therapy (LITT) using laser fibers equipped with an optically diffusive tip and surrounded with a water cooled jacket represent an efficient method for greater control of thermal therapy delivery. By combining LITT with magnetic resonance temperature imaging (MRTI) the evolution of the temperature distribution can be monitored in real time. This can be used in conjunction with gold coated spherical silica core nanoshells or gold coated superparamagnetic iron oxide particles that are tuned to exhibit a plasmon resonance at the optical frequency of the incident laser. This results in increased local absorption and heating, even at low applied laser power. Accurate modeling of the thermal distribution is an essential part of the treatment planning process; not only to predict the 3 dimensional spatial distribution but also how the thermal distribution evolves in time. If the diffusing tip of the fiber was a true line source the thermal distribution would be ellipsoidal in nature. But it has also been demonstrated that the thermal distribution can approximate an ovaloid[1, 2] with the smaller end pointing along the direction of the fiber, this break in ellipsoidal symmetry is due to the directional nature of the photons being transported along the fiber. Both ellipsoidal, ovaloid as well as other coordinates such as limacon are difficult to model in. To alleviate this situation in 2 dimensions the Pennes equation is first solved in circular polar coordinates with appropriate boundary conditions. Conformal mapping is then used to transform the solution into the desired coordinates.

Nanoscale magnetic/luminescent core-shell particles were used for DNA quantification in a hybridization-in-solution format. We demonstrated a simple, high-throughput, and non-PCR based DNA assay for quantifying antibiotic resistance gene tetQ. Fe₃O₄/Eu:Gd₂O₃ nanoparticles (NPs) synthesized by spray pyrolysis were biofunctionalized by passive adsorption of NeutrAvidin. Following immobilization of biotinylated probe DNA on the particles' surfaces, target dsDNA and signaling probe DNA labeled with Cy3 were hybridized with NPs-probe DNA. Hybridized DNA complexes were separated from solution by a magnet, while non-hybridized DNA remained in solution. A linear quantification (R² = 0.99) of a target tetQ gene was achieved based on the normalized fluorescence (Cy3/NPs) of DNANP hybrids. A real-time qPCR assay was used for evaluation of the NPs assay sensitivity and range of quantification. The quantity of antibiotic resistance tetQ genes in activated sludge microcosms, with and without addition of tetracycline or triclosan has been determined, indicating the potential of the optimized assay for monitoring the level of antibiotic resistance in environmental samples. In addition, the tetQ gene copy numbers in microcosms determined by NPhybridization were well correlated with the numbers measured by real-time qPCR assay (R² = 0.92).

Cooled fiber tip technology has significantly improved the volume coverage of laser induced thermal therapy (LITT), making LITT an attractive technology for the minimally invasive treatment of cancer. Gold coated nanoshells can be tuned to experience a plasmon resonance at a desired laser frequency, there introduction into the treatment region can greatly amplify the effectiveness of the thermal treatment. The goal is to conformaly heat the target, while sparing surrounding healthy tissue. To this end a treatment option that is self-confining to the target lesion is highly desirable. This can be achieved in the liver by allowing nanoshells to be taken up by the healthy tissue of the liver as part of their natural removal from the blood stream. The lesion is then incased inside the nanoshell laden tissue of the surrounding healthy tissue. When an interstitial laser probe is introduced into the center of the lesion the thermal radiation scatters outward until it interacts with and is absorbed by the nanoshells located around the lesion periphery. As the periphery heats it acts as secondary source of thermal radiation, sending heat back into lesion and giving rise to ablative temperatures within the lesion while sparing the surrounding tissue. In order to better monitor therapy and know when the target volume has been ablated, or exceeded, accurate knowledge is needed of both the spatial distribution of heating and the maximum temperature achieved. Magnetic resonance temperature imaging (MRTI) is capable of monitoring the spatiotemporal distribution of temperature in vivo[1]. Experiments have been performed in vitro using a dog liver containing nanoshells (concentration 860ppm) and a tissue like lesion phantom designed to have the optical properties of liver metastasis [2].