This PDF file contains the front matter associated with SPIE Proceedings Volume 7674, including the Title Page, Copyright information, Table of Contents, and Conference Committee listing.

Surface-enhanced Raman scattering (SERS) is a powerful tool for intracellular analyses due its minimally invasive nature and molecular specificity. This research focuses on optimizing the sensitivity of SERS in order to widely apply it to the detection of ultra-trace biomolecules within individual living cells. Recent results have shown that large SERS enhancement factors (EF) can be achieved with multi-layer SERS substrates. To fabricate multi-layer SERS substrates, alternating layers of metal films and dielectric spacers are cast over a non-confluent monolayer of nanostructures. Individual particles of these substrates are then immobilized with antibodies to develop SERS-based immuno-nanosensors. The multi-layer SERS EFs can be increased by the use of appropriate dielectric spacer to fabricate the substrates. To further understand the effect of dielectric spacers on the multi-layer SERS EFs, this talk discusses the characterization of the SERS enhancements of multi-layer metal film on nanostructure SERS substrates fabricated with self-assembled monolayer (SAM) spacers. Monolayers with different chain lengths have been systematically capped with varying amount of metal films. It was found that the SERS EFs depend on the nature of the monolayer formed and the amount of metal film deposited on the monolayer. Using optimal SAMs and the appropriate amount of metal film overlayers, SAM multi-layer SERS substrate with optimized SERS intensity can be fabricated. This talk will also explore the functionalization of the SERS substrates with appropriate bio-recognition elements to develop SERS-based immuno-nanosensors.

Since past several years, quantum dot (Qdot) nanotechnology has advanced our capabilities towards sensitive and reliable imaging of biological specimens such as cells, tissues etc. It is well understood that surface passivation is extremely important in obtaining bright and photostable Qdots. Traditionally, an inorganic wide band-gap epitaxially-matched shell material (such as ZnS, ZnSe etc.) is used over the Qdot core (such as CdS, CdSe etc.). Surface passivation minimizes surface related defects and improves quantum efficiency as well as photostability. This is also applicable to dopant based Qdots such as CdS:Mn/ZnS Qdots. In this work we present our research data on CdS:Mn/ZnS Qdots that are further surface modified with appropriate organic ligands for selective sensing applications. These ligands are capable of quenching Qdot luminescence via electron transfer process. Qdot luminescence is restored when electron transfer process is blocked. Using this quenching mechanism, we have demonstrated selective sensing of cadmium ions, alkali metal ions and glutathione in a highly sensitive manner.

Early diagnosis is critical for positive outcome of cancer treatments. In many cases, lives would be saved if the tumor could be detected at a very early stage. Nanoparticles have the property of passively targeting tumor sites due to their enhanced permeation and retention (EPR) effect. Thus they can play a critical role in improving the ability to find cancer in its earliest and most treatable stages. Furthermore magnetic resonance imaging is one of the most precise techniques for cancer screening since it can show 3D images of the tumors. For a better enhancement of the sensitivity of this method, MRI contrast agent (DOTA)Gd was attached to poly(propylene imine) dendrons of third generation and the obtained dendrons were used for modification of gold nanoparticles.

Llama serum contains both conventional IgG as well as unique forms of antibody that contain only heavy chains where antigen binding is mediated through a single variable domain. These variable domains can be expressed recombinantly and are referred to as single domain antibodies (sdAb). SdAb are among the smallest known naturally derived antigen binding fragments, possess good solubility, thermal stability, and can refold after heat and chemical denaturation. Llamas were immunized with either BoNT A or B toxoid and phage display libraries prepared. Single domain antibodies (sdAb) that were able to detect botulinum neurotoxin (BoNT) serotypes A and B were selected from their respective libraries. Here, the binders obtained by panning the BoNT B library on either BoNT B toxoid or BoNT B complex toxoid coated plates or BoNT B toxin coupled microspheres are described. Using these panning methods, we selected for binders that showed specificity for BoNT B. Phage displayed binders were screened, moved to a protein expression vector and soluble sdAb was produced. Using a Luminex flow cytometer binders were evaluated in direct binding assays. We have exploited the unique properties of sdAb and used them as biological recognition elements in immuno-based sensors that can detect BoNT B.

This work describes the rationale behind the preparation of fluorescent probes for imaging lipid peroxyl radicals within membranes of living cells (fluorescent lipophilic antioxidants). The new probes are based on BODIPY dyes tethered to phenol moieties. We discuss the spectroscopic properties of these novel probes, specifically the BODIPY-α-tocopherol analogue B-TOH, and present a molecular level explanation, based on photoinduced electron transfer, that accounts for the significant emission enhancement that the probe BTOH experiences upon reaction with peroxyl free radicals. In addition to the spectroscopy results in homogeneous media, we also describe studies performed in model lipid membranes which show that the sensitivity of BTOH towards lipid peroxyl radicals is somewhat reduced when the probe is membrane embedded. Solutions to increase the sensitivity of the free radical probes are discussed based on the redox potential of BODIPY dyes.

Traditionally micro-well plate based platforms used in biology utilize fluorescence intensity based methods to measure processes of biological relevance. However, fluorescence intensity measurements suffer from calibration drift due to a variety of factors. Photobleaching and self-quenching of the fluorescent dyes cause the intensity signal to drop over the lifetime of sensor immobilized inside the well. Variation in turbidity of the sample during the course of the measurement affects the measured fluorescence intensity. In comparison, fluorescence lifetime measurements are not significantly affected by these factors because fluorescence lifetime is a physico-chemical property of the fluorescent dye. Reliable and inexpensive frequency domain fluorescence lifetime instrumentation platforms are possible because the greater tolerance for optical alignment, and because they can be performed using inexpensive light sources such as LEDs. In this paper we report the development of a frequency domain fluorescence lifetime well-plate platform utilizing an oxygen sensitive transition-metal ligand complex fluorophore with a lifetime in the microsecond range. The fluorescence lifetime dye is incorporated in a polymer matrix and immobilized on the base of micro-well of a 60 well micro-well plate. Respiration measurements are performed in both aqueous and non-aqueous environment. Respirometry measurements were recorded from single Daphnia magna egg in hard water. Daphnia is an aquatic organism, important in environmental toxicology as a standard bioassay and early warning indicator for water quality monitoring. Also respirometry measurements were recorded from Tribolium castaneum eggs, which are common pests in the processed flour industry. These eggs were subjected to mitochondrial electron transport chain inhibitor such as potassium cyanide (KCN) and its effects on egg respiration were measured in real-time.

Most analytical techniques that are routinely used in biomedical research for detection and quantification of biomolecules are time-consuming, expensive and labor-intensive, and there is always a need for rapid, affordable and convenient methods. Recently we have developed a new platform technology for biomolecular detection and analysis: NanoDLSay. NanoDLSay employs antibody-coated gold nanoparticles (GNPs) and dynamic light scattering, and correlates the specific increase in particle size after antigen-antibody interaction to the target antigen concentration. We applied this technology to develop an assay for rapid detection of actin, a protein widely used as a loading control in Western Blot analysis. GNPs were coated with two types of polyclonal anti-actin antibodies, and used in the assay to detect two types of actin: β- and bovine skeletal muscle actin in RIPA buffer. The results of our study revealed some complex aspects of actin binding characteristics, which depended on the type of actin reagent and anti-actin antibody used. A surprising finding was a reverse dose-response relationship between the actin concentration and the average particle size in the assay solution, which we attributed to the effect of RIPA buffer. Our results indicate that RIPA may also interfere in other types of nanoparticle-based assays, and that this interference deserves further study.

The development of new smart sensing methodologies that provide improved sensitivity and/or specificity for rapid and accurate biosensing is highly desirable for in situ and in vivo cancer screening and detection of biological pathogens. However, to date clinical applications of cancer sensing schemes have been for the most part limited by the large patient-to- patient variations in optical response (e.g. fluorescence or Raman signals), as well as by the large variation in background signal levels. A novel biosensing scheme is presented that is based on our recent research showing that the biological matrix may be altered by low intensity (i.e., below the ablation threshold) ultraviolet radiation (primarily 193 to 213 nm) such that the intrinsic fluorescence response is perturbed. Specifically, a sequential combination of optical probing (e.g. fluorescence emission), UV photochemical perturbation, and repeat optical probing is explored as a new spectral dimension based on difference spectroscopy. It is expected that the response is strongly coupled to the local biomolecular matrix. Because the same targeted material is optically probed both before and after perturbation with the UV light source, the resulting differential response may help mitigate variations in the absolute optical response. Preliminary data is presented using imaging mode and spectroscopy modes for several organic matrices as a proof of concept.

A surface plasmon resonance (SPR) sensor to quantify glucose using a molecularly imprinted polymer was developed. The polymer was prepared by crosslinking poly(allylamine) in the presence of glucose phosphate, monobarium salt (GPS-Ba) and attached to a thin film of gold (50 nm) which had been sputtered on top of a glass slide, via amide coupling. Upon removal of the template, this sensor was used to detect glucose in human urine in physiologically significant levels (1-20 mg/ml). Signal enhancement of the glucose sensor was made possible by incorporating gold nanoparticles in the polymer.

The use of Field Programmable Analog Arrays (FPAAs) for analog conditioning of electric potentials on the surface of living tissue, such as Electrocardiography (ECG), is presented in this paper. The inherent reconfigurable capability of these devices provides versatility for circuitry dynamic tuning, i.e., changing amplification gains, filters corner frequencies, signal common levels, etc., without interrupting the signal acquisition path. A study of internal circuitry noise sources is presented and some configurations that attenuate noise effects are proposed.

Due to the narrow vibrational bandwidths and unique molecular fingerprints, Raman spectroscopy can be an information rich transduction technique for chemical imaging. Dynamic systems are often difficult to measure using spontaneous Raman due to the relatively weak scattering cross-sections. Using a Raman enhancement mechanism such as surface enhanced Raman scattering (SERS), exposure times can be reduced to a reasonable level for dynamic imaging, due to the increased Raman signal intensity. This paper will discuss the development of a novel SERS substrate, fabricated on the tips of fiber-optic imaging bundles, which can be integrated into a multispectral imaging system for non-scanning chemical imaging. These substrates are fabricated by mechanically tapering a polished fiber optic imaging bundle consisting of 30,000 individual elements; producing 100-nm or smaller diameter core elements on the distal tip. Chemical etching with hydrofluoric acid creates uniform cladding spikes onto which a SERS active metal is vacuum deposited, forming the SERS active surface. By varying the size of the silver islands deposited on the cladding peaks active, surface plasmons can be tuned to various excitation frequencies. The surface of these tapered fiber optic probes will be evaluated by analysis of the SERS signal, location and shape of the active surface plasmons. The cross talk between the fiber elements will also be evaluated.

Video endoscopy allows physicians to visually inspect inner regions of the human body using a camera and only minimal invasive optical instruments. It has become an every-day routine in clinics all over the world. Recently a technological shift was done to increase the resolution from PAL/NTSC to HDTV. But, despite a vast literature on invivo and in-vitro experiments with multi-spectral point and imaging instruments that suggest that a wealth of information for diagnostic overlays is available in the visible spectrum, the technological evolution from colour to hyper-spectral video endoscopy is overdue. There were two approaches (NBI, OBI) that tried to increase the contrast for a better visualisation by using more than three wavelengths. But controversial discussions about the real benefit of a contrast enhancement alone, motivated a more comprehensive approach using the entire spectrum and pattern recognition algorithms. Up to now the hyper-spectral equipment was too slow to acquire a multi-spectral image stack at reasonable video rates rendering video endoscopy applications impossible. Recently, the availability of fast and versatile tunable filters with switching times below 50 microseconds made an instrumentation for hyper-spectral video endoscopes feasible. This paper describes a demonstrator for hyper-spectral video endoscopy and the results of clinical measurements using this demonstrator for measurements after otolaryngoscopic investigations and thorax surgeries. The application investigated here is the detection of dysplastic tissue, although hyper-spectral video endoscopy is of course not limited to cancer detection. Other applications are the detection of dysplastic tissue or polyps in the colon or the gastrointestinal tract.

Image subtraction has been an extremely useful tool for capturing subtle changes in pixel intensity with extremely high temporal resolution, and has been used for decades in the astronomy and metal corrosion fields. However, to date, image subtraction has not been used as a mainstream technique for investigating morphological changes in cells, tissues, or whole organisms. We introduce a user-friendly differential imaging technique for monitoring real time (~msec) changes in morphology within the micrometer to millimeter spatial scale. The technique is demonstrated by measuring morphological changes morphology for biomedical (bone stress), agricultural (crop root elongation), and environmental (zooplankton ecotoxicology) applications. Subtle changes in growth that would typically only be observed by highly skilled experts are easily resolved via image subtraction and the use of convolution kernels. When coupled with techniques characterizing real time biochemical transport (e.g., respiration, ion/substrate transport), physiology can be directly quantified with a high temporal and spatial resolution. Because of the ease of use, this technique can be readily applied to any field of science concerned with bridging the gap between form and function.

Two novel sensor technologies have been developed for the measurement of skin surface temperature and RF field strength in an RF environment. Such a sensor system would be particularly useful in the test and evaluation of directed energy systems. The sensors operate without being affected by the presence of RF fields and with minimal perturbation of the fields, therefore having a minimal effect on a test. The sensors are designed to be wearable and interface with a portable, battery powered electronics pack by optical fibers. The temperature sensor is based on the variation in fluorescence intensity of a sensor layer with temperature. The RF field sensors operate using a passive circuit that converts the RF field into an optical signal that is measured remotely. Both sensors have been demonstrated in high power microwave lab tests. RF sensor operability has been demonstrated for fields in the range of 0.4 - 8.9 W/cm², while the temperature sensor has been demonstrated over the 30 - 60°C temperature range.

In this work, we present research performed towards the realization of a hypoxia monitor that can detect the onset of hypoxia within a minute with very low false positive and false negative rates. We report the development of the next-generation hypoxia monitor with the capability to simultaneously detect various physiological parameters that change in response to reduced oxygen availability and identify the onset of hypoxia based on the changes in their cross-correlation signals. Significant improvements are obtained over the conventional techniques that are used currently to measure some of the physiological parameters including blood oxygen saturation and blood flow velocity. We demonstrate that a simple patch geometry holding three LEDs and two single photon sensitive detectors can be used to simultaneously obtain the heart rate, respiratory rate, blood flow velocity and blood oxygen saturation levels and in less than one minute analyze their cross-correlation signals to identify the onset of hypoxia from the more benign auto-regulatory response to stress.

We present results of in vitro micro-Raman spectroscopy of normal and cancerous cervical and ovarian tissues excited with 785 nm near-infrared (NIR) laser. Micro- Raman spectra of squamous cervical cells of both cervix and ovarian tissues show significant differences in the spectra of normal and cancerous cells. In particular, several well-defined Raman peaks in the 775-975 cm^-1 region are observed in the spectra of normal cervix squamous cells but are completely missing in the spectra of invasive cervical cancer cells. In the high-frequency 2800-3100 cm^-1 region it is shown that the peak area under CH stretching band is much lower than the corresponding area in the spectra of normal cells. In the case of ovarian tissues, the micro-Raman spectra show noticeable spectral differences between normal cells and ovarian serous cancer cells. In particular, we observed the accumulation of β-carotene in ovarian serous cancer cells compared to normal ovarian cells from women with no ovarian cancer. The NIR micro-Raman spectroscopy offers a potential molecular technique for detecting cervical and ovarian cancer from the respective tissues.

The development of cardiac and pulmonary fibrosis have been associated with overexpression of angiotensin-converting enzyme (ACE). Moreover, ACE inhibitors, such as lisinopril, have shown a benificial effect for patients diagnosed with heart failure or systemic hypertension. Thus targeted imaging of the ACE is of crucial importance for monitoring of the tissue ACE activity as well as the treatment efficacy in heart failure. In this respect, lisinopril-capped gold nanoparticles were prepared to provide a new type of probe for targeted molecular imaging of ACE by tuned K-edge computed tomography (CT) imaging. Concentrated solutions of these modified gold nanoparticles, with a diameter around 16 nm, showed high contrast in CT imaging. These new targeted imaging agents were thus used for in vivo imaging on rat models.

To the casual observer, transient stress results in a variety of physiological changes that can be seen in the face. Although the conditions can be seen visibly, the conditions affect the emissivity and absorption properties of the skin, which imaging spectrometers, commonly referred to as Hyperspectral (HS) cameras, can quantify at every image pixel. The study reported on in this paper, using Hyperspectral cameras, provides a basis for continued study of HS imaging to eventually quantify biometric stress. This study was limited to the visible to near infrared (VNIR) spectral range. Signal processing tools and algorithms have been developed and are described for using HS face data from human subjects. The subjects were placed in psychologically stressful situations and the camera data were analyzed to detect stress through changes in dermal reflectance and emissivity. Results indicate that hyperspectral imaging may potentially serve as a non-invasive tool to measure changes in skin emissivity indicative of a stressful incident. Particular narrow spectral bands in the near-infrared region of the electromagnetic spectrum seem especially important. Further studies need to be performed to determine the optimal spectral bands and to generalize the conclusions. The enormous information available in hyperspectral imaging needs further analysis and more spectral regions need to be exploited. Non-invasive stress detection is a prominent area of research with countless applications for both military and commercial use including border patrol, stand-off interrogation, access control, surveillance, and non-invasive and un-attended patient monitoring.