This PDF file contains the front matter associated with Proceedings of SPIE Volume 6447, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

Successful targeting nanoparticles (NP) to specific cells requires reliable feedback about NP accumulation in cells. This task is a challenge for all optical methods due the size of NP and diffraction limit of optical devices. We modified several microscopy-based techniques for imaging and measuring NP in individual cells: photothermal, fluorescent, electron and atomic-force microscopies and flow cytometry. All those techniques were applied for quantitative analysis and imaging of interaction of gold NP (10 and 30 nm) with living tumor cells. Based on experimental results we performed comparison of all methods in terms of sensitivity, speed, sample requirements etc. We have found that standard microscopes may detect NP and their clusters in individual living cells through imaging NP-related thermal, fluorescent and other phenomena.

The Photon Number-Resolved (PNR) detectors are capable to distinguish between finite numbers n of photons (n = 1, 2, 3, ...) within an ultrafast (femtosecond or picosecond range) week radiation pulse. Recently, a great interest to such devices has been expressed by the quantum information sciences. In the nearest future, an interest to development of such devices can be significantly enhanced by emerging class of new nanophotonic and biological applications, such as single-molecule studies with fluorescent nanoparticles. As it was found recently, the multiexciton state achieved through fast pulse laser irradiation of individual nanoparticle lead to multiphoton character of light emission. The exact knowledge of the number of photons emitted by individual nanoparticle in colloids can provide us with deeper understanding of the interaction between single fluorescent nanoparticle and its environment. However, the development of PNR devices would be necessary to establish such novel photon number-resolved imaging technologies. In this article we briefly review some basic approaches to design and development of the future PNR detectors and electronic readouts.

In this paper, we begin with a brief overview of optical trapping of micro- and nano- particles and of various techniques for the measurement of optical force constants in the linear spring model. We then move on to introduce two complimentary approaches to implement optical forced oscillation of the trapped particle, one by an oscillatory optical tweezers, and the other by chopping (i.e., switching on-and-off) one of the beams in a twin set of optical tweezers. In each implementation, we have measured the steady state amplitude and phase of the oscillating particle as a function of frequency (from ~ 10Hz to 600Hz) with the aid of a quadrant photo-diode in conjunction with a lock-in amplifier. For the case of optical forced oscillation of a "free" particle involving only the optical force and the viscous drag, the experimental data fit fairly well the theoretical curve obtained from the simple linear spring model; both the optical force constant and the viscosity of the surrounding fluid can be deduced with fairly high precision as the fitting parameters from the best fit of the experimental data to the theoretical curves. When one or more external forces, in addition to the optical force and the drag force, were applied to the oscillating particle via mechanisms such as protein-protein interaction or DNA stretching, the oscillating amplitude and phase varied in response to the external forces. Preliminary data showing the change in oscillating amplitude and phase as a function of time in response to external forces will be presented, and potential biomedical applications of this approach will be discussed.

Two-dimensional (2D) phase imaging system based on phase-shift interferometry (PSI) techniques can achieve a very high accuracy, but it has a degraded dynamic characteristic due to the inherent limitation of the PSI. Hence, the phase imaging system is incapable of obtaining real-time information pertaining to phase variations. To develop a label-free, high sensitivity, and dynamic bio-imaging system, a surface plasmon resonance (SPR) biosensing is combined with full-field heterodyne interferometry to develop a common-path full-field heterodyne SPR dynamic phase imager. The phase imager provides some advantages for biosensing such as label-free sensing, high sensitivity, high throughput, long-term stability, and dynamic capability. We build a 16×16 pixel photodiode array with a frame rate of up to 10 kHz as the 2D detector as opposed to a CCD camera with 30 Hz and employ an electro-optic modulator to generate a heterodyne light source. The multi-channel and real-time demodulation is calculated by utilizing a home-made digital signal processing-based lock-in amplifier. The SPR phase imager can detect refractive index changes better than 10^-6 by testing the difference between nitrogen and argon gases, and will be used to analyze the biomolecular interaction on sensing surface with high throughput screening.

The biological compact disc (BioCD) is a sensitive detection platform that detects immobilized biomolecules on the surface of a spinning disc by quadrature laser interferometry. Spinning-disc interferometry (SDI) has the advantage of operating faraway from the 1/f system noise which has a 40 dB per octave slope, thus reducing the detection noise floor by more than 50 dB compared to static interferometric detection techniques. Three quadrature classes of BioCD have been previously reported: micro-diffraction, adaptive optical and phase contrast. In this paper, we introduce a new class of BioCD, the in-line quadrature class, which has achieved a new level of simplicity and sensitivity. A silicon wafer coated by a layer of SiO₂ is used as a substrate for immobilized biomolecules. The thickness of the SiO₂ layer is chosen so that light reflected from the SiO₂ surface on top and the silicon surface below is approximately in phase quadrature. Protein molecules scatter the incident light, adding a phase shift linearly proportional to the mass density of the immobilized protein, which is converted to a far-field intensity shift by quadrature interference. Patterning of protein is achieved by spot printing with a jet printer, which produces protein spots 0.1 mm in diameter. We demonstrate the sensitivity of the in-line quadrature BioCD by an equilibrium dose response experiment on a disc printed with 25,000 proteins spots with a detection limit of 1 ng/mL when divided into 32 virtual wells and treated as 32 separate assays. This current performance is not a fundamental limit, and improvements in disc uniformity will enable scaling up to large numbers of individual assays per disc.

As previously shown the conversion of nucleotide sequences into digital signals offers the possibility to apply signal processing methods for the analysis of genomic data. Genomic Signal Analysis (GSA) has been used to analyze large scale features of DNA sequences, at the scale of whole chromosomes, including both coding and non-coding regions. The striking regularities of genomic signals reveal restrictions in the way nucleotides and pairs of nucleotides are distributed along nucleotide sequences. Structurally, a chromosome appears to be less of a "plain text", corresponding to certain semantic and grammar rules, but more of a "poem", satisfying additional symmetry restrictions that evoke the "rhythm" and "rhyme". Recurrent patterns in nucleotide sequences are reflected in simple mathematical regularities observed in genomic signals. GSA has also been used to track pathogen variability, especially concerning their resistance to drugs. Previous work has been dedicated to the study of HIV-1, Clade F and Avian Flu. The present paper applies GSA methodology to study Mycobacterium tuberculosis (MT) rpoB gene variability, relevant to its resistance to antibiotics. Isolates from 50 Romanian patients have been studied both by rapid LightCycler PCR and by sequencing of a segment of 190-250 nucleotides covering the region of interest. The variability is caused by SNPs occurring at specific sites along the gene strand, as well as by inclusions. Because of the mentioned symmetry restrictions, the GS variations tend to compensate. An important result is that MT can act as a vector for HIV virus, which is able to retrotranscribe its specific genes both into human and MT genomes.

The transport through a modulated energy landscape is a classical problem that, in different forms, arises in different biological systems, such as protein folding and the motion of molecular motors. The calculations become increasingly difficult if a realistic potential landscape is taken into account and in many experimental settings the control of parameters is not possible. We propose and experimentally demonstrate an experimental apparatus that allows direct simulation and imaging of stochastic molecular motion on a time-dependent energy landscape.

Multifunctional nanoparticles hold great promise for drug/gene delivery. Multilayered nanoparticles can act as nanomedical systems with on-board "molecular programming" to accomplish complex multi-step tasks. For example, the targeting process has only begun when the nanosystem has found the correct diseased cell of interest. Then it must pass the cell membrane and avoid enzymatic destruction within the endosomes of the cell. Since the nanosystem is only about one millionth the volume of a human cell, for it to have therapeutic efficacy with its contained package, it must deliver that drug or gene to the appropriate site within the living cell. The successive de-layering of these nanosystems in a controlled fashion allows the system to accomplish operations that would be difficult or impossible to do with even complex single molecules. In addition, portions of the nanosystem may be protected from premature degradation or mistargeting to non-diseased cells. All of these problems remain major obstacles to successful drug delivery with a minimum of deleterious side effects to the patient. This paper describes some of the many components involved in the design of a general platform technology for nanomedical systems. The feasibility of most of these components has been demonstrated by our group and others. But the integration of these interacting sub-components remains a challenge. We highlight four components of this process as examples. Each subcomponent has its own sublevels of complexity. But good nanomedical systems have to be designed/engineered as a full nanomedical system, recognizing the need for the other components.

Diffuse reflectance of cervical tissue treated with gold nanoshells is collected at 0 and 40 degrees, measured from the perpendicular to tissue surface. Between the two collection angles, the infused nanoshells exhibit much stronger scattering signals in contrast to the ordinary reflectance attributed to the naked tissue when the collection angle of the fiber probe is obliquely oriented at 40 degrees. At 0 deg. fiber angle, no significant differentiation is observed between naked and nanoshell-treated tissue samples. This result indicates a strong potential of combining angularly-variable reflectance spectroscopy and gold nanoshells in order to achieve synergistically enhanced scattering contrast in tissue.

We present our first steps towards nanoparticle assisted, optical molecular imaging (NAOMI) using biodegradable nanoparticles. Our focus is on using optical coherence tomography(OCT) as the imaging modality. We propose to use nanoparticles based on biodegradable polymers, loaded with carefully selected dyes as contrast agent, and outline a method for establishing their desired optical properties prior to synthesis. Moreover, we perform a qualitative pilot study using these biodegradable nanoparticles, measuring their optical properties which are found to be in line with theoretical predictions.

Microcantilevers show significant promise in sensing minute quantities of chemical and biological analytes in vapor and liquid media. Much of the reported work on microcantilever sensors has made use of single functionalized microcantilevers, usually derived from commercially available atomic force microscope (AFM) cantilevers. However, arrays with hundreds to thousands of microcantilevers on a single chip are required to create sophisticated, broad spectrum chemical and biological sensors in which individual microcantilevers have different bio- or chemoselective coatings. Unfortunately, the most sensitive microcantilever readout mechanisms (such as laser beam reflection as used in atomic force microscopy) are not readily scalable to large arrays. We therefore introduce a new microcantilever transduction mechanism for silicon-on-insulator (SOI) microcantilevers that is designed to scale to large arrays while maintaining a very compact form factor and high sensitivity. This mechanism is based on in-plane photonic transduction of microcantilever deflection in which the microcantilever itself forms a single mode rib waveguide. Light from the end of the microcantilever is directed across a small gap to an asymmetric receiving waveguide with two outputs that enables differential detection of microcantilever deflection. Initial noise and optical power budget calculations indicate that deflection sensitivities in the 10's of picometer range should be achievable.

Label-free biosensors for protein detection try to overcome the stability and reliability problems of commercialized systems relying on the detection of labeled molecules. We propose a micron sized integrated Silicon-on-Insulator optical biosensor based on a microring cavity that enables real time and sensitive measurements of protein dynamics, fast sample preparation and multiparameter detection for extremely low analyte quantities. Fabrication with Deep UV lithography for standard CMOS processing allows for cheap mass production and integration with electronic functions for complete lab-on-chip devices. The SOI material system offers a high refractive index contrast suitable for the fabrication of submicron sized optical cavities of very high quality. The shift of resonance wavelength that occurs when the dielectric surroundings of such a cavity is changed, is used for sensing. We demonstrate a SOI optical microring resonator with radius 5 micron capable of detecting bulk refractive index changes of 10^-4. Modification of the semiconductor surface, allowing for immobilization of biomolecules, is characterized by X-ray Photoelectron Spectroscopy, ellipsometry, Scanning Contact Angle and Atomic Force Microscopy. We use the avidin/biotin high affinity couple to demonstrate good repeatability and the detection of protein concentrations down to 50 ng/ml. Negative control using low interaction protein couples shows low responses, proving the realization of real specific binding. Integration with a microfluidic setup will allow for more precise monitoring of the interaction dynamics, while lining up the microrings in arrays will allow for cheap high throughput label-free multiparameter analyses.

We have developed a four-channel detection method on a protein-patterned BioCD that simultaneously measures fluorescence, Rayleigh scattering and/or diffraction, and two interferometric channels in orthogonal quadratures: one that measures differential phase and the other that measures direct phase. The latter two channels constitute label-free interferometric protein detection, while fluorescence and Mie scattering detection provide complementary tools. The BioCD is constructed as dielectric coated disks. Protein molecules patterned on dielectric films change the Fresnel reflection coefficient of the films. The change is exhibited in two ways: the reflection coefficient and the phase are both modulated. These are detected simultaneously by a split detector and designated as "amplitude" signal and "phase contrast" (PC) signal. We are able to scan and image patterned proteins across an entire coated disk with high speed in four channels. A single-analyte immunoassay shows strong correlation between the fluorescence channel and the interferometry channel with a detection limit of 10 ng/ml in a complex protein background (rat lysate) concentration of 7 mg/ml.

In this work, we describe our recent efforts aimed at determining the mechanism of signal change for a diffraction-based sensor (DBS) system. The DBS detects analyte-binding events by monitoring the change in diffraction efficiency that takes place when analyte molecules adsorbs to target molecules that have been patterned onto a surface. The exact parameters that affect the intensity of the diffraction intensity are not immediately clear. In this work, it is hypothesized that the intensity of the diffraction signal depends both on the thickness of the diffraction grating and the refractive index of the analyte molecule. This hypothesis has been tested by preparing diffraction grating targets of well-defined thickness from polyelectrolyte multilayers. The index of refraction of the layers was also adjusted by incorporating charged Au nanoparticles into the diffraction grating structure. Results of these experiments are discussed in terms of simple models based on volume-phase holography.

A methodology for enabling biochemical sensing applications using porous polymer photonic bandgap structures is presented. Specifically, we demonstrate an approach to encapsulation of chemical and biological recognition elements within the pores of these structures. This sensing platform is built on our recently demonstrated nanofabrication technique using holographic interferometry of a photo-activated mixture that includes a volatile solvent as well as monomers, photoinitiators, and co-initiators. Evaporation of the solvent after polymerization yields nanoporous polymeric 1D photonic bandgap structures that can be directly integrated into optical sensor systems that we have previously developed. More importantly, these composite structures are simple to fabricate, chromatically tunable, highly versatile, and can be employed as a general template for the encapsulation of biochemical recognition elements. As a specific example of a prototype device, we demonstrate an oxygen (O₂) sensor by encapsulating the fluorophore (tris(4,7-diphenyl-1,10-phenathroline)ruthenium(II) within these nanostructured materials. Finally, we report initial results of extending this technique to the development of a hydrophilic porous polymer photonic bandgap structure for sensing in aqueous environments. The ability to control the hydrophilic/hydrophobic nature of these materials has direct impact on chemical and biological sensing.

We present the optical characterization of an all-dielectric photonic crystal (PC)-based guided resonance filter sensitive to index-of-refraction changes in aqueous solutions. Spectral peak width was found to be 9.8 and 4.4 nm around 816 nm (water media), corresponding to a quality factor Q of 83 and 181, respectively. A spectral shift of peak wavelength with index change of 130 nm/RIU was observed for bulk fluid experiments. Measured peak shift (&Dgr;&lgr;=0.2nm) corresponds to a detectable index change &Dgr;n=1.5×10^-3.

This paper proposes a technique of realizing sub-wavelength focusing spot on surface by modifying the spatial phase in far-field. This focusing spot will use to detect the spectrum of the monolayer biomolecules on the planar surface. Taking the advantage of modifying spatial phase and polarization of incident laser beam, the field distribution in near-field (near focal point) can be changed and can achieve to the smallest spot size of sub-wavelength under the reasonable adjustment of the phase and polarization of incident beam in far-field. Although nano-scale light sources can produce by labelling dye on nanoparticles, quantum dots etc., but technically it is not easy to finely manipulate nanoparticles. On the contrary, using the planar thin film of matured, reliable fabrication processes, not only the near-field twisted electromagnetic of nanostructure can be eliminated, but also fixing the biomolecules on planar surface makes its arrangement to have the consistent direction, thereupon the overall behavior of molecular vibration is simple, pure, and advantageous to detect the vibrational spectra of the monolayer molecules on surface.

We proposed and developed a novel fiber-optic biosensor based on localized surface plasmon coupled emission (LSPCE) which consists of sandwich format of immuno-complex. It is immobilized on the surface of optical fiber where is a fluorescence probe produced by mixing Cy5 labeled antibody and protein A conjugated gold nanoparticles (Au-PA). The fluorophores are excited by localized surface plasmon (LSP) on gold nanoparticle (GNP) surface where the evanescent field is applied near the core surface of unclad optical fiber. Meanwhile, the fluorescence signal is detected by a photomultiplier tube being set beside the unclad optical fiber with high collection efficiency. In the experiment, this novel LSPCE biosensor demonstrates the minimum detectable concentration of mouse immunoglobulin G (IgG) at 1pg/ml (7fM) in the biomolecular interaction with anti-mouse IgG. From the experimental result, it verifies that LSPCE biosensor is a very high sensitive biosensor which is capable of measuring biomolecular interaction at very low concentration.

Previous work with amplitude-sensitive paired surface plasma waves biosensor (PSPWB) demonstrated that the detection sensitivity of PSPWB is able to experimentally detect 0.001% sucrose-water solution and biomolecular interaction of 10pg/ml mouse IgG interacting with immobilized anti-mouse IgG successfully. Further development of the detection sensitivity of PSPWB has been conducted by using 20nm in diameter colloid gold nanoparticles conjugated with target molecules that can result in a higher mass coverage and a larger resonant angle change of plasmon resonance, thereby its detection sensitivity is further enhanced significantly. Bare gold nanoparticles, which is randomly suspended in solution, is adopted to differentiate biospecific binding induced further signal enhancement. Experimentally, the sensitivity at 330fg/ml of Au-nanoparticle conjugated protein A (PA-Au) interacting with mouse IgG which is immobilized on a CM5 sensor chip was detected successfully. By this arrangement, 6-fold signal amplification is demonstrated compared with the same concentration of PA without conjugated gold nanoparticles.