This PDF file contains the front matter associated with SPIE Proceedings Volume 8367, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

We used micro- and resonance Raman spectroscopy with 785 nm and 514.5 nm laser excitation, respectively, to characterize a plant pathogenic bacteria, Xanthomonas axonopodis pv. dieffenbachiae D150. The bacterial genus Xathomonas is closely related to bacterial genus Stenotrophomonas that causes an infection in humans. This study has identified for the first time the unique Raman spectra of the carotenoid-like pigment xanthomonadin of the Xanthomonas strain. Xanthomonadin is a brominated aryl-polyene pigment molecule similar to carotenoids. Further studies were conducted using resonance Raman spectroscopy with 514.5 nm laser excitation on several strains of the bacterial genus Xanthomonas isolated from numerous plants from various geographical locations. The current study revealed that the Raman bands representing the vibrations (v₁, v₂, v₃) of the polyene chain of xanthomonadin are 1003-1005 (v₃), 1135-1138 (v₂), and 1530 (v₁). Overtone bands representing xanthomonadin were identified as 2264-2275 (2v₂), and combinational bands at 2653-2662 (v₁+ v₂). The findings from this study validate our previous finding that the Raman fingerprints of xanthomonadin are unique for the genus Xanthomonas. This facilitates rapid identification (~5 minutes) of Xanthomonas spp. from bacterial culture plates. The xanthomonadin marker is different from Raman markers of many other bacterial genus including Agrobacterium, Bacillus, Clavibacter, Enterobacter, Erwinia, Microbacterium, Paenibacillus, and Ralstonia. This study also identified Xanthomonas spp. from bacterial strains isolated from a diseased wheat sample on a culture plate.

Near-infrared Raman spectroscopy has been used in vitro to identify calcified atherosclerotic plaques in human femoral arteries. Raman techniques allow for the identification of these plaques in a nondestructive manner, which may allow for the diagnosis of coronary artery disease in cardiac patients in the future. As Raman spectroscopy also reveals chemical information about the composition of the arteries, it can also be used as a prognostic tool. The in vivo detection of atherosclerotic plaques at risk for rupture in cardiac patients will enhance treatment methods while improving clinical outcomes for these procedures. Raman spectra were excited by an Invictus 785-nm NIR laser and measured with a fiber-coupled micro-Raman RXN system (Kaiser Optical Systems, Inc., Ann Arbor, MI) equipped with a 785 nm CW laser and CCD detector. Chemical mapping of arteries obtained post mortem allowed for the discrete location of atherosclerotic plaques. Raman peaks at 961 and 1073 cm^-1 reveal the presence of calcium hydroxyapatite and carbonate apatite, which are known to be present in calcified plaques. By mapping the locations of these peaks the boundaries of the plaques can be precisely determined. Areas of varying degrees of calcification were also identified. Because this can be useful in determining the degree of plaque calcification and vessel stenosis, this may have a significant impact on the clinical treatment of atherosclerotic plaques in the future.

We have developed a line-scanning angled fluorescent laminar optical tomography (LS-aFLOT) system. This system enables three-dimensional imaging of fluorescent-labeled stem cell distribution within engineered tissue scaffold over a several-millimeter field-of-view.

It has been shown that non-resonant multiphoton photoacoustic spectroscopy (NMPPAS) has a great potential to be used as a high resolution surgical guidance technique during brain tumor surgery due to its ability of non-invasive or minimally invasive tumor differentiation. However, for experimental purposes associated with method validation, the use of real tissues is not always ideal because of issues such as availability, safety, storage, chemical doping, necessary control of size and shape, etc. To overcome these issues, tissue phantoms made from animal tissues and/or biochemical constituents, are often employed for such analyses. This work demonstrates the ability to develop and characterize gelatin based tissue phantoms with comparable optical and acoustic properties to real tissues by doping the phantoms with a scattering substance, 0.3 μm diameter Al₂O₃ particles. Using these phantoms, light scattering coefficients (μs) of 39 cm^-1 have been generated, which are comparable to real brain tissue, thus making them a great alternative to real tissue for validation studies. In addition, this work also investigates the non-fluorescent species NAD⁺ found in the tissues, to evaluate its potential for being detected by NMPPAS. NMPPAS spectra of NAD⁺ shows a very promising beginning to determine other chemical species such as flavins, collagen, tryptophan, etc responsible for NMPPAS spectral signatures, associated with tumorogenesis.

Narrow-band imaging (NBI) is a spectrally-selective reflectance imaging technique for enhanced visualization of superficial vasculature. Prior clinical studies have indicated NBI's potential for detection of vasculature abnormalities associated with gastrointestinal mucosal neoplasia. While the basic mechanisms behind the increased vessel contrast - hemoglobin absorption and tissue scattering - are known, a quantitative understanding of the effect of tissue and device parameters has not been achieved. In this investigation, we developed and implemented a numerical model of light propagation that simulates NBI reflectance distributions. This was accomplished by incorporating mucosal tissue layers and vessel-like structures in a voxel-based Monte Carlo algorithm. Epithelial and mucosal layers as well as blood vessels were defined using wavelength-specific optical properties. The model was implemented to calculate reflectance distributions and vessel contrast values as a function of vessel depth (0.05 to 0.50 mm) and diameter (0.01 to 0.10 mm). These relationships were determined for NBI wavelengths of 410 nm and 540 nm, as well as broadband illumination common to standard endoscopic imaging. The effects of illumination bandwidth on vessel contrast were also simulated. Our results provide a quantitative analysis of the effect of absorption and scattering on vessel contrast. Additional insights and potential approaches for improving NBI system contrast are discussed.

Small and non-mass-enhancing lesions are diagnostically challenging and easily missed in a routine clinical diagnosis. Compared to mass-enhancing lesions, they show fundamentally di®erent morphologies and kinetic characteristics. To overcome these limitations an automated analysis of such tumors is proposed to determine adequate shape and dynamical descriptors in order to capture this unique behavior. In the present paper, we evaluate several morphological and kinetic features as well combinations of those as potential shape and dynamic descriptors. We will show that for both types of lesions a combination of morphological and kinetic characteristics yields the highest AUC-values compared to dynamic or shape descriptors only. This suggests that for increasing diagnostic accuracy in breast MRI spatio-temporal descriptors for these lesions need to be included in an automated computer-aided system.

Imaging of capillary structures and monitoring of blood flow within vasculature is becoming more common in clinical settings. However, very few dynamic phantoms exist which mimic capillary structures. We report the fabrication and testing of microfluidic, vascular phantoms aimed at the study of blood flow. These phantoms are fabricated using low-cost, off-the-shelf materials and require no lithographic processing, stamping, or embossing. Using laser micromachining, complex microfluidic structures can be fabricated in under an hour. The laser system is capable of producing microfluidic features with sizes on the order of tens of microns, over an area of several square centimeters. Because the laser micromachining system is computer controlled and accepts both vector and raster files, the microfluidic structure can be simple, rectilinear patterns or complex, anatomically correct patterns. The microfluidic devices interface with simple off the shelf syringe pumps. The microfluidic devices fabricated with this technique were used for non-invasive monitoring of flow using speckle based techniques.

There is considerable interest in the development of rapid, point-of-care antibody detection for the diagnosis of infectious and auto-immune diseases. In this paper, we present work on the development of a self-contained microfluidic format for the Luciferase Immunoprecipitation Systems (LIPS) assay. Whereas the majority of immunoassays for antigen-specific antibodies employ either bacteria- or yeast-expressed proteins and require the use of secondary antibodies, the LIPS technique uses a fusion protein comprised of a Renilla luciferase reporter and the antigen of interest produced via mammalian cell culture, ensuring the addition of mammalian post-translational modifications. Patient serum is mixed with the fusion protein and passed over immobilized Protein A/G; after washing, the only remaining luciferase-tagged antigens are those retained by specific antibodies. These can be quantitatively measured using chemiluminescence upon the introduction of coelenterazine. The assay has been successfully employed for a wide variety of diseases in a microwell format. We report on a recent demonstration of rapid HSV-2 diagnosis with the LIPS assay in a microfluidic format, using one microliter of serum and obtaining results in under ten minutes. We will also discuss recent progress on two fronts, both aimed at the deployment of this technology in the field: first, simplifying assay operation through the automation of flow control using power-free means; and second, efforts to increase signal levels, primarily through strategies to increase antibody binding capacity, in order to move towards portable battery powered electronics.

The translation of bio-analytical processes into an automatically functioning microfluidic platform is an attractive task for several reasons. However, due to the complexity of the resulting integrated device covering various process steps like lysis, DNA extraction and purification, continuous-flow PCR and detection etc., these single functional units have to be carefully developed and evaluated in a first step, thus allowing a functional verification prior to final device integration. All the modules as well as the final integrated device have to be manufactured using scalable industrial manufacturing methods, namely injection molding, in order to facilitate commercialization The final integrated device should have a footprint such as SBS-titerplate format, which is generally excepted by the user. For optimal space utilization, microfluidic structures should be on both the top and the bottom side of the device connected with through-holes. The device described in this report is a pathogen DNA analysis example realising all the above prerequisites. Sample is introduced through a Luer-connector in one corner. DNA is extracted in a chamber, which is filled with magnetic beads. All necessary liquid reagents are stored in 500μl modules which are clipped onto the chip. The sample is aliquoted into 8 tracks, liquefies the PCR-reagents which are stored in lyophilized form in small chambers and runs through a meandering channel, in which continuous-flow PCR takes place. The samples are then transferred to the top of the chip and run back to the detection zone where a fluorescence detection of the PCR-products takes place before flowing into waste. As in the device an 8-plex detection is targeted, the spacing of the microchannels after qPCR had to be very narrow in order to have all channels within the field of vision of the detection system.

The world's growing mobility, mass tourism, and the threat of terrorism increase the risk of the fast spread of infectious microorganisms and toxins. Today's procedures for pathogen detection involve complex stationary devices, and are often too time consuming for a rapid and effective response. Therefore a robust and mobile diagnostic system is required. We present a microstructured LabDisk which performs complex biochemical analyses together with a mobile centrifugal microfluidic device which processes the LabDisk. This portable system will allow fully automated and rapid detection of biological threats at the point-of-need.

To combat the threat of biological agents like Yersinia pestis and Brucella melitensis in bioterroristic scenarios requires fast, easy-to-use and safe identification systems. In this study we describe a system for rapid amplification of specific genetic markers for the identification of Yersinia pestis and Brucella melitensis. Using chip based PCR and continuous flow technology we were able to amplify the targets simultaneously with a 2-step reaction profile within 20 minutes. The subsequent analysis of amplified fragments by standard gel electrophoresis requires another 45 minutes. We were able to detect both pathogens within 75 minutes being much faster than most other nucleic acid amplification technologies.

Preventing bacterial contaminations is a significant challenge in applications across a variety of industries, e.g. in food processing, the life sciences or biohazard detection. Here we present a fully automated lab-on-a-chip system wherein a disposable microfluidic chip moulded by polymeric injection is inserted into an operating device. Liquid samples, here obtained from an air sampler, can be processed to extract and lyse bacteria, and subsequently to purify their DNA using a silica matrix. After the washing and elution steps, the DNA solution is dispensed into a reaction vessel for further analysis in a conventional laboratory polymerase chain reaction (PCR) device. We demonstrate the workability and efficiency of our approach with results from a 9 ml liquid sample spiked with E. coli.

Rapid, accurate, and minimally-invasive biosensors for glucose measurement have the potential to enhance management of diabetes mellitus and improve patient outcome in intensive care settings. Recent studies have indicated that implantable biosensors based on Förster Resonance Energy Transfer (FRET) can provide high sensitivity in quantifying glucose concentrations. However, standard approaches for determining the potential for interference from other biological constituents have not been established. The aim of this work was to design and optimize a FRET-based glucose sensor and assess its specificity to glucose. A sensor based on competitive binding between concanavalin A and dextran, labeled with long-wavelength acceptor and donor fluorophores, was developed. This process included optimization of dextran molecular weight and donor concentration, acceptor to donor ratio, and hydrogel concentration, as well as the number of polymer layers for encapsulation. The biosensor performance was characterized in terms of its response to clinically relevant glucose concentrations. The potential for interference and the development of test methods to evaluate this effect were studied using a potential clinical interferent, maltose. Results indicated that our biosensor had a prediction accuracy of better than 11% and that the robustness to maltose was highly dependent on glucose level.