This PDF file contains the front matter associated with SPIE Proceedings Volume 8954, including the Title Page, Copyright information, Table of Contents, and Conference Committee listing.

A technique was developed to achieve enhanced coherent anti-Stokes Raman scattering (CARS) imaging using selfassembled silica microspheres. In this study, a layer of optically transparent silica microspheres was self-assembled onto polymer grating samples to enhance the CARS signals. The highest enhancement of 12.5 was achieved using 6.1-μmdiameter microspheres for C-H molecule vibration. Finite-difference time-domain (FDTD) algorithm under the perfectly matched layer boundary condition was used to simulate the enhancement using silica microspheres of different diameters.

We developed a two-dimensional multispectral imager that exploits the extraordinary optical transmission of nanohole arrays (NHAs) in a metal film for color separation. The NHA device consisted of blocks in a tiled arrangement, where each block contained multiple metallic NHAs, each with unique feature geometry to enable separation of a mixture of colors into distinct spectral bands. The NHA device was integrated into an optical imaging system. The system was used to capture near video-rate images of a scene and then unmix the colors within the scene into 4 spectral bands in the nearinfrared regime.

Surface Enhanced Raman Spectroscopy is a powerful analytical technique that combines the excellent chemical specificity of Raman spectroscopy with the good sensitivity provided by the enhancement of the signal observed when a molecule is located on (or very close to) the surface of suitable nanostructured metallic materials. The availability of cheap, reliable and easy to use SERS substrates would pave the road to the development of bioanalytical tests that can be used in clinical practice. SERS, in fact, is expected to provide not only higher sensitivity and specificity, but also the simultaneous and markedly improved detection of several targets at the same time with higher speed compared to the conventional analytical methods. Here, we present the SERS activity of 2-D plasmonic crystals made by polymeric pillars embedded in a gold matrix obtained through the combination of soft-lithography and plasma deposition techniques on a transparent substrates. The use of a transparent support material allowed us to perform SERS detection from support side opening the possibility to use these substrates in combination with microfluidic devices. In order to demonstrate the potentialities for bioanalytical applications, we used our SERS active gold surface to detect the oxidation product of apomorphine, a well-known drug molecule used in Parkinson’s disease which has been demonstrated being difficult to study by traditional HPLC based approaches.

Nanopore-based sensing is an attractive candidate for developing single-molecule DNA sequencing technology. Recently, optical detection with a parallel nanopore array has been demonstrated. Although this method is a promising approach to develop high thorough-put measurement, the approach requires observation at low-background condition. In this paper, we propose a new optical method for nanopore DNA sequencing with high resolution and a high signal-tonoise ratio. We use ultraviolet light for the excitation of a fluorescent probe and a nanopore in a silicon membrane. Because silicon has a large refractive index and an extinction coefficient at ultraviolet wavelengths, light transmission thorough the membrane is negligible. This contributes to low background measurement of fluorescence from fluorophore-labeled DNA strands. In addition, the z-polarization component of the electric field is attributed to generating a large electric field gradient at the nanopore exit due to its boundary condition at the silicon surface. Our numerical electromagnetic simulation revealed that the z-component electric field was dominant compared to the xcomponent electric filed. The intensity of the electric field increased steeply in 2 nm, when ultraviolet light of 375nm wavelength was focused on a 10nm-thick silicon membrane with a 7 nm-diameter nanopore. This steeply increasing electric field can be sufficient resolution for the sequencing of designed DNA polymer. Finally, our experimental results demonstrated optical detection of single DNA translocation events with a high signal-to-noise ratio under applied voltage.

During cancer radiotherapy protocols, the early profile of energy deposition is decisive for the prediction and control of radiation-induced biomolecular and sub-cellular damage. A major challenge of spatio-temporal radiation biomedicine, a newly emerging interdisciplinary domain, concerns the complete understanding of biophysical events triggered by an initial energy deposition inside confined ionization clusters (tracks) and evolving over several orders of magnitude, typically from femtosecond (1 fs = 10^-15 s) and sub-nanometer scales. The innovating advent of femtosecond laser sources providing ultra-short photon beam and relativistic electron bunches, in the eV and MeV domain respectively, open exciting opportunities for a real-time imaging of radiation-induced biomolecular alterations in nanoscopic tracks. Using a very short-lived quantum probe (2p-like excited electron) and high-time resolved laser spectroscopic methods in the near IR and the temporal window 500 – 5000 fs, we demonstrate that short-range coherent interactions between the quantum probe and a small biosensor of 20 atoms (disulfide molecule) are characterized by an effective reaction radius of 9.6 ± 0.2 angströms. For the first time, femtobioradical investigations performed with aqueous environments give correlated information on spatial and temporal biomolecular damages triggered by a very short lived quantum scalpel whose the gyration radius is around 6 angströms. This innovating approach would be applied to more complex biological architectures such as nucleosomes, healthy and tumour cells. In the framework of high-quality ultra-short penetrating radiation beams devoted to pulsed radiotherapy of cancers, this concept would foreshadow the development of real-time nanobiodosimetry combined to highly-selective targeted pro-drug activation.

The progress in stem cell research over the past decade holds promise and potential to address many unmet clinical therapeutic needs. Tracking stem cell with modern imaging modalities are critically needed for optimizing stem cell therapy, which offers insight into various underlying biological processes such as cell migration, engraftment, homing, differentiation, and functions etc. In this study we report the feasibility of photothermal optical coherence tomography (PT-OCT) to image human mesenchymal stem cells (hMSCs) labeled with single-walled carbon nanotubes (SWNTs) for in vitro cell tracking in three dimensional scaffolds. PT-OCT is a functional extension of conventional OCT with extended capability of localized detection of absorbing targets from scattering background to provide depth-resolved molecular contrast imaging. A 91 kHz line rate, spectral domain PT-OCT system at 1310nm was developed to detect the photothermal signal generated by 800nm excitation laser. In general, MSCs do not have obvious optical absorption properties and cannot be directly visualized using PT-OCT imaging. However, the optical absorption properties of hMSCs can me modified by labeling with SWNTs. Using this approach, MSC were labeled with SWNT and the cell distribution imaged in a 3D polymer scaffold using PT-OCT.

Super-resolution fluorescence microscopy is anticipated to be a powerful tool in observing biological structures and processes smaller than the diffraction limit of light microscopy (~200nm). Yet, many super-resolution techniques (STORM/PALM, STED) employ photo-switchable fluorescent probes (i.e., dyes and fluorescent proteins) that are limited in brightness and stability, reducing potential image resolution. Here, we describe photo-switchable quantum dots (QDs) with enhanced brightness and stability, and excellent optical properties, including narrow emission spectra and broad excitation spectra, compared to fluorescent dyes. These QDs are composed of one green QD, one gold nanoparticle (AuNP), and complimentary single stranded DNA (ssDNA) modified with photo-sensitive azobenzene groups bound to each of the particles. Because of the azobenzene photosensitive property, the ssDNA strands hybridize when excited with visible light, yielding a QD-AuNP conjugate in which QD fluorescence is quenched through Förster resonance energy transfer (FRET); and dehybridize under visible light, yielding separate QDs and AuNPs that are free to diffuse from each other. Because FRET is strongly distance dependent (i.e., α 1/r⁶, in this case, a few nanometers), QD fluorescence is restored. Moreover, the photo-switchable QD-AuNP conjugate scheme has the potential to be integrated with a DNA nano-machine platform, adding the potential for photo-manipulated functionality. As a preliminary proof of concept, we tethered different nanocomponents, including QD micelle assemblies and AuNPs, to DNA origami structures (hinge and platform shapes) using ssDNA hybridization.

Localized surface plasmon enhanced microscopy based on nanoislands of random spatial distribution was demonstrated for imaging live cells and molecular interactions. Nanoislands were produced without lithography by high temperature annealing under various processing conditions. The localization of near-field distribution that is associated with localized surface plasmon on metallic random nanoislands was analyzed theoretically and experimentally in comparison with periodic nanostructures. For experimental validation in live cell imaging, mouse macrophage-like cell line stained with Alexa Fluor 488 was prepared on nanoislands. The results suggest the possibility of attaining the imaging resolution on the order of 80 nm.

Modern routine enzyme immunoassays for detection and quantification of biomolecules have several disadvantages such as high cost, insufficient sensitivity, complexity and long-term execution. The surface plasmon resonance of silver nanoparticles gives reasons of creating new in the basis of simple, highly sensitive and low cost colorimetric assays that can be applied to the detection of small molecules, DNA, proteins and pollutants. The main aim of the study was the improving of enzyme immunoassay for detection and quantification of the target molecules using silver nanoparticles. For this purpose we developed method for synthesis of silver nanoparticles with hyaluronic acid and studied possibility of use these nanoparticles in direct determination of target molecules concentration (in particular proteins) and for improving of enzyme immunoassay. As model we used conventional enzyme immunoassays for determination of progesterone and estradiol concentration. We obtained the possibility to produce silver nanoparticles with hyaluronan homogeneous in size between 10 and 12 nm, soluble and stable in water during long term of storage using modified procedure of silver nanoparticles synthesis. New method allows to obtain silver nanoparticles with strong optical properties at the higher concentrations – 60-90 μg/ml with the peak of absorbance at the wavelength 400 nm. Therefore surface plasmon resonance of silver nanoparticles with hyaluronan and ultraviolet-visible spectroscopy provide an opportunity for rapid determination of target molecules concentration (especial protein). We used silver nanoparticles as enzyme carriers and signal enhancers. Our preliminary data show that silver nanoparticles increased absorbance of samples that allows improving upper limit of determination of estradiol and progesterone concentration.

We propose a novel detection method based on the symmetry breaking induced by the bio-molecule to be detected. Briefly, by choosing a sensor presenting a particular symmetry, the revolution symmetry, the adsorption of an analyte will break this symmetry. By detecting this change in the symmetries of the system, the presence of bio-molecules can be detected. This optical method provides substantial advantages over current approaches for the conception of biosensors. In particular, this approach relies on geometrical considerations, providing important properties such as the possibility to multiplex spatially or in wavelength. In addition, it relaxes strongly the constrains on the sensor as no specific plasmon resonances are necessary. We believe this work opens promising alternatives for the development of biosensors.

Respiratory mucus is one of the main barriers for nanoparticle-based pulmonary delivery systems. This holds true especially for lung diseases like cystic fibrosis, where a very tenacious thick mucus layer hinders particle diffusion to the lung epithelium or the target area. Typically, mean square displacement of particles is used for mobility evaluation. In contrast, our objective is to develop a feasible technique to track directed particle penetration as a prerequisite for efficient pulmonary nanotherapy. Therefore, particle diffusion in artificial mucus was monitored based on confocal laser scanning microscopy (CLSM) and particle-mucus interaction was observed. As pharmaceutical relevant and benign materials, solid lipid nanoparticles (SLNs) were prepared by hot-melt emulsification using glyceryl behenate and different stabilizing agents such as poloxamer-407, tween-80, and polyvinyl alcohol (PVA). The diffusion of labeled SLNs in stained artificial sputum representing CF-patient sputum was verified by 3D time laps imaging. Thus, the effect of coating, particle size and mucus viscosity on nanoparticle diffusion was studied. Using image analysis software "Image J", the total fluorescent signal after 30 min in case of poloxamer-coated SLNs was 5 and 100 folds higher than tween- and PVA-coated SLNs, respectively. Nevertheless, increasing mucus viscosity reduced the diffusion of tweencoated SLNs by a factor of 10. Studying particle-mucus interaction by CLSM can be considered a promising and versatile technique.

Silicon-on-insulator microring resonators have proven to be an excellent platform for label-free nanophotonic biosensors. The high index contrast of the silicon-an-insulator platform allows for fabrication of micrometer size sensors and a high degree of multiplexing. To enable robust, low-noise performance of a microring resonator sensor chip in a lab-on-a-chip setting, flood illuminating an array of vertical grating couplers is a promising approach to couple input light into the chip. This technique provides a very high alignment tolerance while at the same time exciting multiple sensors simultaneously for rapid parallel read-out. We demonstrate this technique to obtain a highly multiplexed chip output combined with real time sensor information. However, parasitic reflections on the chip surface can deteriorate the sensor signal and limit the performance. We investigate the use of surface structures to limit these parasitic signals and show a significant improvement of the sensor operation.

Fluorescence readout of molecular information is a promising approach for biomolecular sensing. For detection of enormous biomolecules via uorescence, biomolecular information should be converted to codes that can be readout easily and simultaneously. For the purpose, we study a biomolecule uorescence color (B/F) encoders that modulate uorescence signals by control of uorescence resonance energy transfer (FRET). The B/F encoder converts biomolecular signals into uorescent color codes represented with uorescent wavelengths and intensity levels. The combination offers a great number of codes for representing the biomolecular information. In this study, we discuss multiplexed detection of target biomolecules using B/F encoders. Use of the B/F encoders would offer a multiplexed biomolecular sensing in a one-pot without micro-fabrication like DNA microarray. In the experiments, we prepared B/F encoders based on two kinds of hybridization chain reactions (HCR) that make long double-stranded DNA polymers to control positions of uorescence and quencher molecules. In the B/F encoders, target molecules trigger to start assembling the polymer structures. The uorescent molecules in the absence of the targets are near the quenchers and the output uorescence is suppressed by FRET. The polymerization process separates the uorescent and quencher dyes and the uorescent signal increase. The experimental results show that the B/F encoders based on HCRs have linear and independent response to each target, and temporal signals during the encoding reactions are usable for multiplexed readout. This result leads to the multiplexed sensing in a one-pot by uorescent ampli cation and multiple uorescent color-coding.

We present a polymer optical waveguide integration technology for the detection of nanoparticles in an evanescent field based biosensor. In the proposed biosensor concept, super-paramagnetic nanoparticles are used as optical contrast labels. The nanoparticles capture target molecules from a sample fluid and bind to the sensor surface with biological specificity. The surface-bound nanoparticles are then detected using frustration of an evanescent field. In the current paper we elaborate on the polymer waveguides which are used to generate a well-defined optical field for nanoparticle detection.

Diagnosis of Phenylketonuria (PKU) in newborns is important because it can potentially help prevent mental retardation since it is treatable by dietary means. PKU results in phenylketonurics having phenylalanine levels as high as 2 mM whereas the normal upper limit in healthy newborns is 120 uM. To this end, we are developing a microfluidic platform integrated with a SERS substrate for detection of high levels of phenylalanine. We have successfully demonstrated SERS detection of phenylalanine using various SERS substrates fabricated using nanosphere lithography, which exhibit high levels of field enhancement. We show detection of SERS at clinically relevant levels.

In this article we describe the fabrication of free standing n-type porous silicon microcavity (MC) and their properties as liquid sensors. We have optimized the etching recipe to keep both large pore size and high quality factor (Q-factor). Thus the fabricated porous layers have pore size in the range of 40 to 110 nm and are thus compatible with mass transport across the porous layer. We found that MC with a Q-factor of 60 can measure down to 1.1*10^-5 refractive index variations. Furthermore we analyze the role of non specific binding by comparing flow through versus flow over geometries. We compare these two approaches using different techniques and we show that flow over assay systematically overestimates the sensitivity of the device because of an inefficient rinse of the sample. Our work clearly indicates a limit in the reliability of measurements performed in flow over geometry unless specific controls are taken into account.

We have investigated near field distribution and absorption cross-section of CdSe quantum dot (QD) conjugated gold nanoparticles using three-dimensional finite-difference time-domain method. A gold nanoparticle core was modeled with a different SiO2 shell thickness and surface density of QDs. Absorption cross-section was found to be proportionate to the shell thickness and the QD surface density. In regard to the absorption by a single QD, either nanoparticle-QD coupling or QD-QD coupling was dominant depending on the surface density. Moreover, shell thickness weakens the coupling and the absorption of a single QD. Finally, enhanced absorption by a QD-conjugated nanoparticle dimer structure is also reported as a result of field enhancement between NPs assisted by QDs.

Despite the fact, that a range of clinically viable imaging modalities, such as magnetic resonance imaging (MRI), computed tomography (CT), photo emission tomography (PET), ultrasound and bioluminescence imaging are being optimised to track cells in vivo, many of these techniques are subject to limitations such as the levels of contrast agent required, toxic effects of radiotracers, photo attenuation of tissue and backscatter. With the advent of nanotechnology, nanoprobes are leading the charge to overcome these limitations. In particular, single wall nanotubes (SWNT) have been shown to be taken up by cells and as such are effective nanoprobes for cell imaging. Consequently, the main aim of this research is to employ mesenchymal stem cells (MSC) containing SWNT nanoprobes to image cell distribution in a 3D scaffold for cartilage repair. To this end, MSC were cultured in the presence of 32μg/ml SWNT in cell culture medium (αMEM, 10% FBS, 1% penicillin/streptomycin) for 24 hours. Upon confirmation of cell viability, the MSC containing SWNT were encapsulated in hyaluronic acid gels and loaded on polylactic acid polycaprolactone scaffolds. After 28 days in complete chondrogenic medium, with medium changes every 2 days, chondrogenesis was confirmed by the presence of glycosaminoglycan. Moreover, using photothermal optical coherence tomography (PT-OCT), the cells were seen to be distributed through the scaffold with high resolution. In summary, these data reveal that MSC containing SWNT nanoprobes in combination with PT-OCT offer an exciting opportunity for stem cell tracking in vitro for assessing seeding scaffolds and in vivo for determining biodistribution.

The video for this presentation is unavailable.