This PDF file contains the front matter associated with SPIE Proceedings Volume 7908, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

We propose a new method of local illumination based on the activation of glass substrates, enables us to drive fluorescence investigations on biological sample at nanometric scale. This local fluorescence excitation is achieved through a non-radiative energy transfer. We demonstrate the potentiality of our technique through two biological relevant applications: imaging of the cell adhesion points and fluorescence correlation spectroscopy in a sub-diffracted volume (attoliter range).

We present a model based on Mie Surface Double Interaction (MSDI) to explore bead-based detection mechanisms using imaging and scanning. The application goal of this work is to explore the trade-offs between the sensitivity and throughput among various detection methods. Experimentally we use thermal oxide on silicon to establish and control surface interferometric conditions. Surface-captured gold beads are detected using Molecular Interferometric Imaging (MI2) and Spinning-Disc Interferometry (SDI).

The corral trap is a novel, microscale device for user-controlled trapping of single fluorescent molecules and other nanoand microparticles in solution. This trapping method does not require particle monitoring for trap operation, has multiparticle trapping capabilities, and can be turned off at will to release the trapped particle(s). We present an improved fabrication method and describe the integration of the corral trap into a microfluidic device, which will further increase its versatility and expand its range of applications.

Magnetic resonance imaging (MRI) is one of the best imaging modalities for noninvasive cancer detection but MRI does not have enough sensitivity to delineate tumor margins during surgery. Moreover, since most surgical tools contain metal substances, image-guided surgery is hard to perform with a MR machine using magnets. Also, MR imaging is too slow for real-time guided-surgery. On the other hand, near infrared fluorescence (NIRF) imaging has recently received great interest for in vivo imaging due to its high signal-to-noise ratios and short image-acquisition times. NIRF imaging can be used to delineate tumor margins during surgery, but current NIRF imaging cannot provide the penetration depth to detect early-stage cancer inside body. Thus, we have developed dual-modality in vivo imaging for MRI detection of tumors and NIRF-guided surgery using multi-component nanoparticles. NIRF dye (cyanine 5.5, Cy5.5), conjugated glycol chitosan nanoparticles (HGC) exhibited excellent tumor targeting ability with NIRF imaging. Superparamagnetic iron oxide (SPIO) nanoparticles as a MR contrast agent were loaded into the nanoparticles, resulting in SPIO-HGC-Cy5.5 nanoparticles. SPIO-HGC-Cy5.5 nanoparticles were characterized and evaluated in mice by both NIRF and MR imaging. Our results indicate SPIO-HGC-Cy5.5 nanoparticles have the potential for dual-modality in vivo imaging with MRI detection of tumors and NIRF-guided surgery.

A single exposure of digital Gabor holography (DGH) is used for simultaneous three-dimensional measurement of particle position. The particle sample is set up such that its position can be electro-mechanically manipulated using calibrated piezoelectric transducers in both the lateral and axial directions. The central position of the reconstructed image of the particle is determined by low-pass filtering, thresholding, and center-of-mass calculation. We have obtained less than 20 nm resolution in both the lateral and axial directions in a direct and unambiguous manner. The method is applied to calibration of optical trap strength.

Vibrational spectroscopy has been used to elucidate the temperature dependence of structural and conformational changes in lipids and liposomes. In this work, the thermal properties of lipid-based nanovesicles originating from a newly developed self-forming synthetic PEGylated lipids has been investigated by variable-temperature Fourier-transform infrared (FTIR) absorption and Raman spectroscopic methods. Thermally-induced changes in infrared and Raman spectra of these artificial lipid based nanovesicles composed of 1,2-dimyristoyl-rac-glycerol-3-dodecaethylene glycol (GDM-12) and 1,2-distearoyl-rac-glycerol-3-triicosaethylene glycol (GDS-23) were acquired by using a thin layered FTIR spectrometer in conjunction with a unique custom built temperature-controlled demountable liquid cell and variable-temperature controlled Raman microscope, respectively. The lipids under consideration have long hydrophobic acyl chains and contain various units of hydrophilic polyethylene glycol headgroups. In contrast to conventional phospholipids, this new kind of lipid is forming liposomes or nanovesicles spontaneously upon hydration, without supplying external activation energy. We have found that the thermal stability of such PEGylated lipids and nanovesicles differs greatly depending upon the acyl chain-lengths as well as associated head group units. However, the thermal behavior observed from both spectroscopic vibrational techniques are in good agreement.

Solvent effects were studied in fluorescence resonance energy transfer (FRET) from a cationic polyfluorene copolymer (FHQ, FPQ) to a fluorescein (Fl)-labeled oligonucleotide (ssDNA-Fl). Upon addition of dimethyl sulfoxide (DMSO), optical properties of the polymers and the probe dye were substantially modified. And the FRET-induced Fl emission was measured by directly exciting the polymer within the complex, polymer/ssDNA-Fl. The FRET signal was successfully modulated with changing the DMSO content. In the case of FHQ, the FRET-induced Fl emission was seriously quenched in phosphate buffer solution (PBS), while a salient FRET signal was observed in a 80 vol% DMSO/PBS mixture (36.8 time higher than that in PBS). The FPQ-sensitized FRET signal was also 3.8-fold amplified by the presence of DMSO. That result is from the decrease of hydrophobic interactions between the polymer and ssDNA-Fl, which induces the weaker polymer/ssDNA-Fl complexation with longer intermolecular separation. The gradual decrease in Fl PL quenching with increasing the DMSO content was investigated by measuring the Stern- Volmer quenching constants (3.3-4.2 × 10⁶ M^-1 in PBS, 0.56-1.1 x 10⁶ M^-1 in 80 vol% DMSO) in PBS/DMSO mixtures. The substantially reduced PL quenching would amplify the resulting FRET Fl signal. This approach suggests a simple way of modifying the fine-structure of polymer/ssDNA-Fl and improving the detection sensitivity in conjugated polymer-based FRET bioassays.

While extremely relevant to many life science fields, such as biomedical diagnostics and drug delivery, studies on the size of nanoparticulate matter dispersed in biofluids are missing due to a lack of suitable methods. Here we report that fluorescence single particle tracking (fSPT) with maximum entropy analysis is the first technique suited for accurate sizing of nanoparticles dispersed in biofluids, such as whole blood. After a thorough validation, the fSPT sizing method was applied to liposomes that have been under investigation for decades as nanocarriers for drugs. The tendency of these liposomes to form aggregates in whole blood was tested in vitro and in vivo. In addition, we have demonstrated that the fSPT sizing technique can be used for identifying and sizing natural cell-derived microparticles directly in plasma. fSPT sizing opens up the possibility to systematically study the size and aggregation of endogenous or exogenous nanoparticles in biofluids.

Various allotropes of Carbon nanoparticles (CNP) are emerging as very important building blocks for nanotechnology and biomedical applications due to their unique electronic, optical, mechanical and thermal properties. We report synthesis of crystalline CNPs from benzene using electric plasma discharge method under controlled laboratory environment. With varied electric field, different allotropes of carbon were synthesized as observed under high resolution electron microscope and selected area electron diffraction, optical spectroscopic studies revealed distinct differences between these CNPs. Raman spectroscopy of these CNPs showed a distinct peak at 1330 cm^-1 (characteristic of defect band) and another peak at 1600 cm^-1 (graphitic band). The ratio of defect to graphitic band was found to increase with increasing voltage between Fe-electrodes. Further, the ratio was altered when CNPs were formed using graphite-electrodes. Fluorescence spectroscopic measurements showed evident blue fluorescence exhibited by CNPs formed at relatively higher voltage between two Fe-electrodes. This was attributed to the increasing Fe-content, as measured by Energy dispersive X-ray analysis (EDX) and vibrating sample magnetometer (VSM). Addition of exogenous dyes in benzene during synthesis of CNPs using electric plasma discharge led to formation of fluorescent nanotubes. These fluorescent CNPs can be functionalized to target cancer cells for both imaging and targeted photothermal therapy using near-IR laser beam.

Barium titanate (BaTiO₃) is a good candidate for phase conjugation on the nano-scale, as four-wave mixing has been shown in nanoparticles of this type. Also, the ability to dope this material with rare earth elements, with strong absorption and emission lines, makes it possible to use these as multi-functional, multi-modal probes for biomedical applications. BaTiO₃ nanoparticles are synthesized using a precipitation method and fully characterized. These particles are used in a four wave mixing setup to optically conjugate scattered light traveling through turbid media, such as tissue, to re-obtain lost image information due to the scattering process.

Paclitaxel interferes with the normal function of microtubule breakdown, induces apoptosis in cancer cells and sequesters free tubulin. As this drug acts also on other cell mechanisms it is important to monitor its accumulation in the cell compartments. The intracellular spreading of the drug was followed using a WITEC 300R confocal Raman microscope equipped with a CCD camera. Hence Atomic force microscopy (an MFP3D- Asylum Research AFM) in imaging and force mode was used to determine the morphological and mechanical modifications induced on living cells. These studies were performed on living epithelial MCF-7 breast cancer cells. Paclitaxel was added to cell culture media for 3, 6 and 9 hours. Among the specific paclitaxel Raman bands we selected the one at 1670 cm^-1 because it is not superposed by the spectrum of the cells. Confocal Raman images are formed by monitoring this band, the NH₂ and the PO₄ band. Paclitaxel slightly accumulates in the nucleus forming patches. The drug is also concentrated in the vicinity of the cell membrane and in an area close to the nucleus where proteins accumulate. Our AFM images reveal that the treated cancerous MCF-7 cells keep the same size as the non treated ones, but their shape becomes more oval. Cell's elasticity is also modified: a difference of 2 kPa in the Young Modulus characterizes the treated MCF-7 mammary cancerous cell. Our observations demonstrate that paclitaxel acts not only on microtubules but accumulates also in other cell compartments (nucleus) where microtubules are absent.

Here, we report in situ formation of microstructures from the regular constituents of culture media near live cells using spatially-structured near infrared (NIR) laser beam. Irradiation with the continuous wave (cw) NIR laser microbeam for few seconds onto the regular cell culture media containing fetal bovine serum resulted in accumulation of dense material inside the media as evidenced by phase contrast microscopy. The time to form the phase dense material was found to depend on the laser beam power. Switching off the laser beam led to diffusion of phase dark material. However, the proteins could be stitched together by use of carbon nanoparticles and continuous wave (cw) Ti: Sapphire laser beam. Further, by use of spatially-structured beam profiles different structures near live cells could be formed. The microfabricated structure could be held by the Gravito-optical trap and repositioned by movement of the sample stage. Orientation of these microstructures was achieved by rotating the elliptical laser beam profile. Thus, multiple microstructures were formed and organized near live cells. This method would enable study of response of cells/axons to the immediate physical hindrance provided by such structure formation and also eliminate the biocompatibility requirement posed on artificial microstructure materials.

The miniaturization of nanostructure-based optical sensing devices requires integration of multiple nanostructure patterns with smaller spacing. However, the effect of plasmonic cross-talk and its effect on spectral transmission are not fully understood. In this paper, we experimentally fabricated different sets of nano-hole arrays (each with an area of 30 μm x 30 μm) of various hole diameter, hole spacing, and inter-array spacing. The spectral transmission of each nano-hole array was measured and the effect of inter-array spacing on peak transmission and resonance wavelength was determined.

The paper presents an image-oriented modality to functionally describe artificially and biologically nanostructured surfaces, which can be used for the characterization of the atom neighborhoods on the surface of proteins. The property which is mainly analyzed in this paper is the hydrophobicity distribution on protein surface, but the distributions of charges and mutual electrical potentials can also be considered. The actual discrete hydrophobicity distribution attached to the atoms that form a surface atom's vicinity is replaced by an approximately equivalent hydrophobicity density distribution, computed in a standardized octagonal pattern around each atom. These representation of hydrophobicities is used to compute the resemblance of surface atom neighborhoods belonging to a protein, defined as the sum of the products of hydrophobicity densities of the corresponding patches (the pattern's central circles or angular sectors having the same position). The similitude and the interaction of a pair of atom neighborhoods are defined as their resemblance for parallel, respectively, anti-parallel orientations of the normals on the molecular surfaces in the points where the central atoms are located. The purpose of this work is to create a database of selected protein surfaces that will be used for nanotechnology research and applications purposes.

In recent years two-photon photopolymerization has emerged as a novel and extremely powerful technique of three-dimensional nanostructure formation. Complex-shaped structures can be generated using appropriate beam steering or nanopositioning systems. Here, we report on the fabrication of three-dimensional arrangements made of biocompatible polymer material, which can be used as templates for cell growth. Using three-dimensional cell cages as cell culture substrates is advantageous, as cells may develop in a more natural environment as compared to conventional planar growth methods. The two-photon fabrication experiments were carried out on a commercial microscope setup. Sub-15 fs pulsed Ti:Sapphire laser light (centre wavelength 800 nm, bandwidth 120 nm, repetition rate 85 MHz) was focused into the polymer material by a high-numerical aperture oil immersion objective. Due to the high peak intensities picojoule pulse energies in the focal spot are sufficient to polymerize the material at sub-100 nm structural element dimensions. Therefore, cell cages of sophisticated architecture can be constructed involving very fine features which take into account the specific needs of various types of cells. Ultimately, our research aims at three-dimensional assemblies of photopolymerized structural elements involving sub-100 nm features, which provide cell culture substrates far superior to those currently existing.

Pillar-array based optical cavities have unique properties, e.g., having a large and connected low dielectric index space (normally air space), having a large percent of electric field energy in air and standing on a substrate. These properties make them well suitable to make ultra compact and highly sensitive label-free optical sensors to detect bio-/chemical reactions. We designed, fabricated, and measured a silicon-on-insulator pillar array microcavity that possesses a quality factor as high as 27,600. We studied its sensitivity for both bulk index change and surface index modification. As a bulk index sensor, for environmental refractive index change of 0.01, a resonance peak wavelength shift of 3.5 nm was measured. As a surface index sensor, the simulations show, for a coating with thickness of 1 nm, the resonance wavelength shifts as large as 2.86 nm. Combining with a sharp 0.06 nm wide resonance peak, our pillar-array sensor is able to resolve ultra small bulk and surface refractive index changes caused by target molecules.

High throughput plasmonic sensors are a popular research field, standard surface plasmon resonance (SPR) instruments can achieve high throughput only in imaging configuration. This leads to consideration of pattern substrates and isolated nanoparticle arrays, both of which have some disadvantages. Spot functionalisation relies upon mask or pin printing to accomplish density, and this increase the complexity of use and standard operating procedures. Both patterned and nanoparticle arrays assay platforms are also commonly single use, unlike some SPR imaging and multi channel angular sensing SPR approaches. The microarray format proposed here is intended for multiple usages and regenerated, with a simple optical readout method. A plasmonic polymer of exquisite refractive index sensitivity and incorporate glass-like physical and mechanical stability provides the sensing element to the platform. Further, the standard sol-gel chemistry is well understood and amenable to easy covalent functionalisation as well as matrix methods such as nitrocellulose for biomolecule fictionalization. Two forms of polymer templating have been developed. For spots greater than 700μm a double side tape method can be applied and for sub 700μm patterned SU-8 and 100nm Aluminum reflective layer allow greater spot resolution. Proof of concept through refractive index sensing is demonstrated.

A nanoarray based-single molecule detection system was developed for detecting proteins with extremely high sensitivity. The nanoarray was able to effectively trap nanoparticles conjugated with biological sample into nanowells by integrating with an electrophoretic particle entrapment system (EPES). The nanoarray/EPES is superior to other biosensor using immunoassays in terms of saving the amounts of biological solution and enhancing kinetics of antibody binding due to reduced steric hindrance from the neighboring biological molecules. The nanoarray patterned onto a layer of PMMA and LOL on conductive and transparent indium tin oxide (ITO)-glass slide by using e-beam lithography. The suspension of 500 nm-fluorescent (green emission)-carboxylated polystyrene (PS) particles coated with protein-A followed by BDE 47 polyclonal antibody was added to the chip that was connected to the positive voltage. The droplet was covered by another ITO-coated-glass slide and connected to a ground terminal. After trapping the particles into the nanowells, the solution of different concentrations of anti-rabbit- IgG labeled with Alexa 532 was added for an immunoassay. A single molecule detection system could quantify the anti-rabbit IgG down to atto-mole level by counting photons emitted from the fluorescent dye bound to a single nanoparticle in a nanowell.

Digital Holographic Microscopy produces quantitative phase analysis of a specimen with excellent optical precision. In the current study, this imaging method has been used to measure induced thermal lensing by optical excitation in the time-resolved regime with excellent agreement to model predictions. We have found that the thermal effect should not be dismissed when pursuing optical radiation pressure experiments, even when the media involved are transparent. We have developed a unified model and simulated methods of decoupling the two effects. The results of this study and simulations suggest that our near term goal of nanometric measurement of an optical pressure induced deformation will prove successful. Precise measurement of this phenomenon can be useful in determining physical properties of interfacial surfaces, such as surface tension, and characterizing physical properties of cellular structures.

Numerous studies provide evidence that nanosecond electric pulses (nsEPs) can trigger the formation of nanopores in the plasma membranes of cells. However, the biophysical mechanism responsible for nanopore formation is not well understood. In this study, we hypothesize that membrane damage induced by nsEPs is primarily dependent on the local molecular composition and mechanical strength of the plasma membrane. To test this hypothesis, we positioned metal-coated, nanoscale cantilever tips using an atomic force microscope (AFM) to deliver nsEPs to localized areas on the surface of the plasma membrane. We conducted computational modeling simulations to verify that the electric field provided by the nsEP is concentrated between the tip and the plasma membrane. The results show that we could effectively deliver nsEPs using the AFM tips at very low voltages. Using scanning electron microscopy we analyzed the tips after applying 10V over 5 seconds duration and found no damage to the tip or loss of platinum coating. As a proof of concept, we applied a 1 and 10V, 5 second pulse to HeLa cells causing large morphological changes. We also applied both a mechanical indention and 600ns electrical pulse stimulus and measured positive propidium ion uptake into the cytoplasm suggesting formation of membrane pores. In future studies, we plan to elucidate the effect that specific, local molecular structures and compositions have on efficacy of electroporation using the newly constructed nano-electrode system.

We present the design principles and performance characteristics of a prototype user-friendly shear force based Tip-Enhanced Raman Spectroscopy system. High numerical aperture reflective optics are utilized to optimize photon delivery and collection, while allowing for interrogation of samples with varying resistivities, thicknesses, and opacities. The integration of tips and tuning forks into manufacturable units is investigated to facilitate simple tip replacement. Finally we discuss methods to mitigate the remaining challenges to the technique becoming routine and user-friendly.

In this study, a few optical immunosensors were developed to determine the concentration of BMP-7. Hydrophilic CdSe/ZnS quantum dots (QDs) were synthesized and conjugated to the antibody of BMP-7 (BMP-7Ab). The QDconjugated BMP-7Ab was used as a fluorescence probe at excitation and emission wavelengths of 470 nm and 585 nm, respectively. It was immobilized either on the bottom of the well of a 96-well microtiter plate or on the tip of an optical fiber. Two immunoassays, i.e. the direct and sandwich assays, were studied for their sensitivity. The sensitivity of the direct immunoassay was 1296.21, compared to 384.69 for the sandwich assay. The linear detection range was 0.0-1.0 ng/mL for both assays. Based on the results of the microtiter plate technique, the direct assay technique was used for the development of an optical fiber immunosensor. The optical fiber immunosensor has a linear detection range between 0.0 and 10.0 ng/mL with a detection limit of 0.413 ng/mL. The optical fiber immunosensor was applied to the sequential injection analysis for the automatic determination of BMP-7.

This paper presents a novel, high sensitive and miniaturized fluorescence detection system which integrated a LED light source, all necessary optical components and a photodiode with preamplifier into one package about 2 cm x 2 cm x 2 cm especially for the applications of lab-on-a-chip, portable bio-detection system and point-of-care diagnostic system. The prototype has been tested using the fluorescence dye 5-Carboxyfluorescein (5-FAM) dissolved into solvent DMSO (Dimethyl Sulfoxide) and diluted with DI water as the testing solution samples. Resolution approximation method is accepted to evaluate the sensitivity. The testing results prove a remarkable sensitivity at pico-scale molar, around 1.08 pM/L, which should meet the most of bio-detection requirements. This cost-effective detection system can be widely integrated to the portable device and system for fluorescent detection in biological, chemical, medical, point-of-care applications.