This PDF contains front matter associated with SPIE Proceedings Volume 8025, including Title Page, Copyright Information, Table of Contents, and the Conference Committee listing.

Surface-enhanced Raman scattering (SERS) has become an attractive analytical tool for intracellular analyses due to its minimally invasive nature and molecular specificity. However, highly reproducible and optimized SERS substrates have been seen as a key to developing SERS as a reliable analytical methodology. This research focuses on optimizing self assembled monolayer (SAM)-based multilayer SERS substrates for a wide range of applications, including ultratrace detection of biomolecules within individual living cells. Multilayer SERS substrates are comprised of alternating layers of metal film and dielectric spacer cast on a monolayer of nanostructures. Using these substrates, varying degrees of SERS enhancement factors (EF) have been achieved, some as large as 10-fold relative to optimized single film over nanostructures substrates. To gain a mechanistic understanding of multilayered SERS enhancements, SAMs have been used to systematically vary spacer thickness. The results revealed spacer-dependent SERS EFs. To further the understanding of multilayer SERS enhancement, this work discusses the use of terminating functional groups in the optimization of SAM multilayer SERS substrates. SAMs having various functional groups were used as dielectric spacers to systematically vary the dielectric constant. To investigate the effect of the pH on the uniformity of the SAMs and their multilayer SERS enhancement, SAMs were formed in alkylthiol solutions of different pH and the subsequent SERS enhancement were evaluated. It was found that using alklythiol SAMs with appropriate terminating functional groups the SAM multilayer can achieve SERS EFs ranging between 10⁸ and10¹⁰ and the substrates yielded highly reproducible SERS signals. The effect of the pH on the SERS enhancement is selective on the type of the terminating functional group of the alkylthiol used for SAM formation.

We have developed a simple and potentially a low-cost method for the sensitive detection of target proteins via surface-enhanced Raman scattering (SERS). The immunosensor constructed by the conjugation of monoclonal antibodies to 20 nm diameter gold nanoparticles via the bifunctional Raman reporter molecule, 5, 5'dithiobis (succinimidyl-2-nitrobenzoate) (DSNB) is the basis of a membrane-bound detection system. Traditionally, a common laboratory technique called a dot blot, which is a colorimetric method where detection of proteins is accomplished through the application of assorted dyes followed by their measurement via a densitometer. Dot blotting is a convenient and time saving method that involves the spotting of a protein onto an immobilizing matrix, such as nitrocellulose (NC) or polyvinylidene fluoride (PVDF) membrane. We found that for detection via SERS spectroscopy NC is the matrix of choice because it offers low background, minimal preparation prior to protein application, and optimal position of Raman bands. Furthermore, SERS detection of protein on NC requires only minimal sample preparation and demonstrates increased sensitivity when compared to other dot blot detection methods. Depending on the dye used for visualization, dot blots analyzed by commonly used optical methods have limits of detection in the nanogram range, some as low as 20 pg/ml. Here we demonstrate the use of the dot blot method for detecting target proteins (e.g., protein A and prostate specific protein (PSA)) by SERS spectroscopy down to a concentration of 100 fg/ml.

Autonomous technologies are needed which are capable of sensing real time changes in biophysical transport across cell membranes/organelles. These technologies must not only be highly sensitive/selective, but must also be minimally invasive/intrusive, causing no significant physical/chemical effects on cell behavior. Challenges with mainstream technologies (e.g., assays, fluorescent dyes, microsensors) include signal noise/drift, low temporal resolution, requirement of large sample sizes, cytoxicity, organelle sequestration, and intracellular buffering. Recent advancements in fiber optics have greatly enhanced the performance of microsensors (e.g., increased sensitivity/selectivity, response time), but used in concentration mode near cells/tissues these sensors suffer from poor signal to noise ratio. Work over the last few decades has advanced microsensor utility through sensing modalities that extend and enhance the data recorded by sensors. This technique, known as self-referencing, converts static micro/nanosensors with otherwise low signal-to-noise ratios into dynamic flux sensors capable of filtering out signals not associated with active transport by acquisition and amplification of differential signals. Here, we demonstrate the use of a self-referencing referencing frequency domain fiber optic microsensor containing a quenched dye (platinum tetrakis-pentafluorophenyl porphyrin) for quantifying cell/tissue flux in biomedical, agricultural, and environmental applications.

Surface-enhanced Raman scattering (SERS) utilizing colloidal silver and gold has been demonstrated to provide a rapid means of measuring the Raman spectra of microorganisms in the fingerprint region. In this study, we have introduced microcavity substrates coated with alternating layers of silver and gold thin films for measuring the Raman spectra of four strains of E. coli. These microcavitiy substrates have been prepared by placing glass microspheres between two polished aluminum substrates and pressing them together using a standard lab press. After removing the glass microspheres from the substrates, the substrates have been coated with 15 to 70 nm thick films of chromium, silver and gold in a precise order. The cavities were evaluated for SERS enhancement by measuring Raman spectra of dilute rhodamine 6G (R6G) down to 10^-8 M. With these microcavities, we have investigated the SERS spectra of four chemically competent strains of E. coli (One Shot OmniMAX 2-T1, Mach1-T1, Stbl3, and TOP10). Replicate SERS spectra of all the four e-coli strains show excellent reproducibility. Visual examination of the spectra, however, reveals differences in the spectra of these strains. To confirm this observation, we have used multivariate analysis for positive identification and discrimination between the strains.

Several new techniques are emerging to use digital display devices as new type of light source for analytical purpose. We demonstrate the use of a liquid crystal display (LCD) computer screen as excitation light source for determining gaseous oxygen contents over a relatively large area. Meso-scale device platforms are prepared with glass plates incorporating fluidic channels and commercial oxygen sensor patches. Fluorophores are excited by blue wavelength range from the LCD computer screen. The sensor signals exhibit a good linear relationship with respect to the oxygen content. The display devices, with a capability of uniform illumination over a large area for variable wavelengths, have a great potential as light source for high-throughput, multiple-analyte, quantitative chemical analysis.

Raman optical activity (ROA) spectroscopy is a chiral sensitive vibrational spectroscopic technique that measures the difference of molecular responses to either left or right circularly polarized photon radiation. As a structural sensitive probe, ROA has great potential applications in fabricating bio-molecular sensors due to its capability of capturing small changes in molecular structure. However, ROA is a weak effect on the order of 10^-3 or 10^-4 smaller than its parent spontaneous Raman. Extension of ROA to widely sensing applications can be accelerated by combining it with surface plasmon resonance effect, surface-enhanced Raman spectroscopy (SERS), allowing for trace chemical detections. However, it has been shown to be very challenging to combine SERS with ROA to obtain a reliable surface-enhanced Raman optical activity measurement by several independent ROA research groups. This is because of the complexity of interaction of chiral molecules with metal surfaces. In addition, another main factor for the difficulty is the lack of a depolarized ROA spectrometer which can remove effectively huge polarized Raman bands, generally the major sources of artifacts in ROA. In this paper, we will discuss the development of a depolarized Raman spectrometer. This newly developed depolarized Raman instrument is based on the BioTools' ChiralRAMAN-2X scattered circular polarization (SCP) ROA spectrometer and has been modified into a dual circular polarization ROA spectrometer with the introducing circularity conversion in the incident radiation path. Combing the simultaneous detection of left and right circularly polarized scattering photons, newly developed Raman instrument are capable of modulate both incident and scattered light simultaneously resulting dual circular polarization (DCP) ROA measurements from depolarized parent Raman scattering. The performance of the Raman spectrometer has been initially evaluated with standard sample of small molecules for the proof-of-principle measurements of DCP-ROA, and its potential application in SEROA measurement has also been discussed.

We developed a simple oxygen imaging platform with phosphorescent oxygen sensor films to demonstrate a quantitative oxygen determination method utilizing a color CCD camera. Phosphorescence quenching of a luminophore Pt(II) meso-tetrakis (pentafluorophenyl) porphyrin complex (PtTFPP) immobilized in poly (dimethylsiloxane) (PDMS) matrix, is the principal detection mechanism. This sensor material was cast to form a film on the bottom surface of a transparent Petri dish. As levels of dissolved oxygen increased, phosphorescence of the complex decreased, allowing for measurement of oxygen levels which developed in the sensor film. A camera with a charge-coupled device (CCD) was used in conjunction with processing software to quantify oxygen levels colorimetrically. Microscopic images were collected using a CCD camera and stored as a set of red/green/blue (RGB) images. Phosphorescence excitation (390 nm peak) is limited to the blue (B) pixels of the CCD chip, and these values were discarded; while retaining the oxygen-responsive phosphorescence emission (645 nm peak) almost identical with the response range of the red (R) pixels. Red pixel intensity analysis effectively extracts color intensity information, which can be in turn directly related to oxygen contents. Color CCD cameras allow simultaneous acquisition of many types of chemical information by combining the merits of digital imaging with the attributes of spectroscopic measurement. Therefore, use of color CCD cameras is considered as an inexpensive alternative to time-resolved imaging for relatively short-term monitoring.

Micro- Raman spectroscopic investigation of ALVAC virus and of normal chicken embryo fibroblast cells and the cells infected with ALVAC virus labeled with green fluorescence protein (GFP) were performed with a 785 nm laser. Good quality Micro-Raman spectra of the Alvac II virus were obtained. These spectra show that the ALVAC II virus contains buried tyrosine residues and the coat protein of the virus has α-helical structure. A comparison of Raman spectra of normal and virus infected chicken embryo fibroblast cells revealed that the virus infected cells show additional bands at 535, 928, and 1091 cm^-1, respectively, corresponding to δ(C-O-C) glycosidic ring, protein α-helix, and DNA (O-P-O) modes. In addition, the tyrosine resonance double (833 and 855 cm^-1) shows reversal in the intensity of the higher-frequency band as compared to the normal cells that can be used to identify the infected cells. In the C-H stretching region, the infected cells show bands with higher intensity as compared to that of the corresponding bands in the normal cells. We also found that the presence of GFP does not affect the Raman spectra of samples when using a 785 nm micro-Raman system because the green fluorescence wavelength of GFP is well below the Stokes-Raman shifted spectral region.

Raman spectra of anti-HIV-1 antibody, HIV-1 antigen (p24), and HIV-1 antibody-antigen complex have been measured in near-infrared and UV regions: 785 nm; 830 nm; and 244 nm laser excitations. The spectrum of the HIV-1 antigen was excited with an infrared laser and contains numerous Raman peaks. The most prominent peaks are broad bands at 1343, 1449, 1609 and 1655 cm^-1, which are characteristic of the Raman spectra of biological cells. The UV Raman spectrum of the HIV-1 antigen has a completely different structure. It has two strong peaks at 1613 cm^-1 and 1173 cm^-1. The peak at 1613 cm^-1 is associated with vibrations of the aromatic amino acids tyrosine (Tyr) and tryptophan (Try). The second strongest peak at 1173 cm^-1 is associated with the vibration of Tyr. The Raman peak pattern of the HIV-1 antigen-antibody complex is very similar to that of the HIV-1 antigen. The only difference is that the peak at 1007 cm^-1 of the Raman spectrum of the HIV-1 antigen-antibody complex is slightly enhanced compared to that of the HIV-1 antigen. This indicates that the peaks of the HIV-1 antigen dominate the Raman spectrum of the HIV-1 antigen-antibody complex.

Photoacoustic spectroscopy is a powerful optical biopsy technique that enables rapid tumor diagnosis in situ. It has also been reported that photoacoustic spectroscopy can be used to diagnose pre-malignant tissue based on the chemical differences between healthy and pre-malignant tissues. Since the acoustic signals obtained from tissues in these analyses suffer from minimum damping, photoacoustic spectroscopy can be highly sensitive. This paper focuses on the characterization of a novel multiphoton excited photoacoustic methodology for margining of malignant and pre-malignant tissues. The two-photon excitation process in tissues using nanosecond laser pulses produces ultrasonic signals that transmit through tissue with minimal attenuation. Additionally, the two-photon excitation process is highly localized since only ballistic photons contribute to the excitation process; thereby eliminating potential absorption events in tissue not of interest (i.e., along the beam path) and increasing the spatial resolution of the diagnostic technique to that achievable via optics. This work characterizes the two-photon excitation process for photoacoustic signal measurements on a model dye. Using gelatin phantoms to mimic real tissues, tissue penetration studies were performed, revealing chemical species as deep as 1.3 cm in the tissue can easily be detected using this methodology. Furthermore, the resolution of this multiphoton excitation process was determined to be as great as 50 μm (near cellular level resolution).

In cancer detection, imaging techniques have a great importance in early diagnosis. The more sensitive the imaging technique and the earlier the tumor can be detected. Contrast agents have the capability to increase the sensitivity in imaging techniques such as magnetic resonance imaging (MRI). Until now, gadolinium-based contrast agents are mainly used for MRI, and show good enhancement. But improvement is needed for detection of smaller tumors at the earliest stage possible. The dendrons complexed with Gd(DOTA) were synthesized and evaluated as a new MRI contrast agent. The longitudinal and transverse relaxation effects were tested and compared with commercial drug Magnevist, Gd(DTPA).

In vivo experiments were performed to measure temperature elevations in prostatic tumors implanted in mice during laser photothermal therapy using nanorods. Ti:Sapphire laser tuned at 800 nm irradiating the surface of the tumor delivered an average laser power of 0.5 - 1.0 W, equivalent to a laser radiance of 1.3-2.6 W/cm². The temperature elevations measured by two thermocouples located at the center and bottom of the tumor have shown non-uniform tumor temperature field, 10-30°C above the baseline temperature of 37°C. The experimental studies have demonstrated the capability of confining laser energy in tumors by a very small amount of nanorod solution.

For patients with a history of heart attack or stroke, the prevention of another cardiovascular or cerebrovascular event is crucial. The development of cardiac and pulmonary fibrosis has been associated with overexpression of tissue angiotensin-converting enzyme (ACE). Recently, gold nanoparticles (GNPs) have shown great potential as X-ray computed tomography (CT) contrast agents. Since lisinopril is an ACE inhibitor, it has been used as coating on GNPs for targeted imaging of tissue ACE in prevention of fibrosis. Herein, lisinopril-capped gold nanoparticles (LIS-GNPs) were synthesized up to a concentration of 55 mgAu/mL. Their contrast was measured using CT and the results were compared to Omnipaque, a commonly used iodine-based contrast agent. The targeting ability of these LIS-GNPs was also assessed.

Bio-stimulation by a light is called the Light Therapy (LT) and an Infrared Illuminator (IRI) provides the human sleeping in a Healing Bed (HB) continuously a dose of the IR radiation. General specifications for the IRI given below and its data are discussed in the paper. (1) Use of 60" wide by 80" long queen size bed. (2) A LED providing 1.5 mw at 1550 nm is selected. (3) 60 LED are mounted in series and parallel on a chip and this chip is mounted on top of the HB.

The anti-sleepiness sensor systems have been devised for soldier's sober mental condition. These systems judge whether the soldier is sleepy or not, on one hand by monitoring open or closed eyes, on the other hand by measuring the heart blood beat and rate on the carotid of human's neck. They reasonably adopt one of the following methods such as optical, mechanical, magnetic impedance and piezoelectric sensor and so on. In this paper, the characteristics of those sensors are compared to one another and subsequently the suitable ones are proposed from the viewpoint of measurement and judgment reliability.; as a sensor to directly monitor the soldier's open/closed eyes the IR (Infrared) sensor is recommended, which is equipped on glasses (so called the anti-sleepiness glasses), and as a sensor to measure the heart beat and rate of blood vein, the piezoelectric PMN-PT crystal sensor mounted on a necklace turns out to be the most suitable owing to its high sensitivity (i.e. the anti-sleepiness necklace). These systems and relevant ideas are also applicable to the civilian usage, namely to the student preparing an examination as well as to the car-driver for safety.

In this paper, we introduce a novel surface acoustic wave (SAW) based two-stage microfluidic platform for continuous particle focusing and separation. The driving force is acoustic radiation force generated by surface acoustic waves in a microfluidic device over a piezoelectric substrate. The prominent features of this two-stage sorter are that particle focusing and separation are accomplished simultaneously and the sorter doesn't require the use of the sheath flow for positioning or aligning of particles, thereby decreasing the complexity. Moreover, no physical contact is needed between the ultrasonic transducer and the medium. The ultrasonic treatment has not been shown to damage cells or biological material as well. Prior to separation, the particles are lined up to the center of the channel in the first stage without adding any sheath flow. After passing the first stage, the larger particles move to the pressure node more quickly than the smaller particles according to the difference of the acoustic forces acting on them. As the acoustic force is proportional to the volume of the particles, the larger particles are subjected to larger acoustic force. Consequently, the flow focused particles are separated with different lateral displacement along the cross-section of the channel. In the present paper, we discuss our design and fabrication efforts as well as review state-of-the-art particles manipulation techniques that are currently employed.