Our aim is to use nanocrystals (NC) to study endothelial cell biology, in particular, the cell surface receptor Tie2. Tie2 is highly expressed in endothelial cells and is critical for angiogenesis and vascular maintenance. Conjugating NCs to the Tie2 ligands, the angiopoietins, and tracking their characteristic luminescence lines will allow us to study the regulation of Tie2 in vitro and in vivo. To study NC behavior in a relevant biological system, endothelial cells were grown and cultured in vitro. Two different types of NC were made and tested: two-color core-shell CdSe/ZnS and more complicated nanostructures containing also Au clusters. Measurements were performed in specially prepared media with different pH values, as well as in the cell growth medium. The positions of NC-related luminescence lines were not influenced by the kind of media which makes them suitable for biolabeling the cell surface receptor Tie2. At the same time, the relative magnitude of the NC peaks depends on the pH of the medium and can therefore be used for characterization of the latter. Confocal microscopic images show that NCs with different ligands demonstrate different distributions inside living cells.

The interaction between semiconductor nanocrystals (quantum dots) and biological structures and cells is strongly influenced by nanoparticle size, exposure to light, and surface cap or conjugate. Hydrophobic particles can insert into lipid bilayers, often resulting in membrane leakage. Oxidizing and reducing agents can photosensitize the quantum dot, yielding greater potential for cell damage; however, penetration into cells is seen only if the particle is specifically targeted to a receptor or antigen on the cell. When particles interact with DNA, oxidation can occur as measured by the presence of hydroxyguanine, preventing cellular replication. In this paper, several cell-free and whole-cell systems are presented to investigate the mechanisms for nanoparticle entry across lipid bilayers, the evolution of their surface composition with light and oxygen exposure, and their potential as targeted cytotoxic drugs and/or environmental hazards. Uptake into mammalian cells, Gram positive and Gram negative bacteria were compared and contrasted in order to identify important factors in nanoparticle uptake and toxicity. Simultaneously, Fourier transform infrared spectroscopy (FTIR) and time-correlated single photon counting (TCSPC) were used to track quantum dot surface degradation with time. Finally, a lipid bilayer system was used to investigate nanoparticle-membrane interactions. The advantages of this system are that its composition is fully known, so that the role of cell-surface receptors is eliminated, and recordings may be performed in the dark. These studies allowed for the formulation of preliminary models of quantum dot binding and entry that consider novel variables.

Photoluminescent porous Si (pSi) is a potentially attractive material for biosensor devices. Its ease of fabrication, large active surface area and unique optical properties are just some important attributes. Among other transduction techniques, it is possible to monitor the onset of molecular binding events through the effective quenching of the bright pSi photoluminescence. Here we present the study of effective quenching through a colloidal Ag nanoparticle interaction with pSi. Placing the metallic nanoparticles in close proximity to the light emitting pSi can effectively sweep away the charge carriers from the semiconductor surface and result in a carrier depletion region near the Si-nanoparticle interface. By labeling the targeted bio-species with a silver nanoparticle, and the pSi surface with an appropriate receptor molecule ; in-situ PL monitoring can provide a real-time transduction scheme for the pSi- based biosensor devices.

Femtosecond laser ablation of a gold plate in aqueous solutions has been used to form colloidal gold nanoparticles. Using different chemical environments during the fabrication, this method makes it possible to functionalize nanomaterials by an appropriate capping ligand. In particular, we were able to control the size and the reactivity of gold particles by using different polymers (dextran and polyethylene glycol). The size of the nanoparticles, measured by transmission electron microscopy (TEM), was found to be as low as 5 nm, in some cases. The addition of these capping agents also significantly improved the long term stability of gold particles. The produced nanoparticles exhibited a strong absorption band near 520 nm due to the surface plasmon resonance and a photoluminescence signal in the violet-blue spectral range. The functionalized nanoparticles produced are of significance in view of their bio-imaging and bio-sensing applications.

The novel mechanical and electrical properties of carbon nanotubes have potential applications in a variety of fields, and their size makes them appealing for use in biosensors. However, in order to exploit their potential, carbon nanotubes must be functionalized for biologic compatibility. Sulfonated and polyanilinated multi-walled and single-walled carbon nanotubes were investigated by several spectroscopic and microscopic methods to asses their feasibility in characterization of functionalized carbon nanotubes (CNT). Transmission electron microscopy (TEM), electron energy loss spectroscopy (EELS) and energy-dispersive X-ray analysis (EDX) were applied to confirm the presence of heteroatoms that were not present in the parent CNTs. EELS ratio mapping, used in conjunction with bright-field TEM imaging, is an extremely powerful tool for examining functionalized nanotubes and other composites because it provides a means of showing the spatial arrangement of individual elements in a sample. This characterization approach provides clear evidence of functional group incorporation onto the CNT. Subsequent atom mapping along the vicinity of the tube structure also allowed us to illustrate the three dimensional distribution of the heteroatoms along the CNT surface. Other elemental analysis techniques can confirm and possibly quantify the presence of a particular element, but fail to illustrate its spatial distribution in a material. In this study, the presence of nitrogen and sulfur atoms along the surface of a nanotube have been confirmed and shown to be uniform by means of TEM and EELS. EDX plots have been investigated as an additional measure of functionalization.

Fullerene derivatives have appealing properties that can potentially be used in materials science and medical applications. In particular, fullerenes are known to produce reactive oxygen species upon their excitation with light. This makes them particularly attractive as photosensitizers for photodynamic therapy (PDT). Photodynamic therapy is a new modality of treatment of cancer as well as some non-cancerous conditions. It involves the combined actions of a drug (photosensitizer) and light to produce a cytotoxic effect. Water-soluble hexa(sulfo-n-butyl)[60]fullerenes (FC₄S) was reported recently to generate singlet oxygen (¹O₂) and superoxide radical (O₂^-·) upon its excitation with light, making it a promising candidate for PDT treatments. Recently, we synthesized new amphiphilic fullerene derivatives, namely, [60]fullerene-diphenylaminofluorene-oligo(ethylene glycol) conjugates, C₆₀(>DPAF-PEG600) and C₆₀(>DPAF-PEG2000), as potential photosensitizers. In this paper we compare FC₄S to PEG-based fullerenes in terms of their singlet oxygen photosensitization ability. We measured time-resolved kinetics of singlet oxygen luminescence photosensitized by excitation of fullerenes via a 10 ns pulsed laser at 523 nm. For FC₄S we observed "normal" kinetics with a monoexponential decay profile giving a time constant 3.8 us in water. In contrast, for the case of C₆₀(>DPAF-PEG600) and C₆₀(>DPAF-PEG2000), a non-monoexponential decay profile with a long tail (~ 10² μs) in water was observed. We hypothesize that this is due to formation of vesicles by PEG fullerenes in aqueous solution. To investigate photodynamic activity of these fullerene derivatives in vitro, we used HeLa human adenocarcinoma and B16 mouse melanoma cell lines. FC₄S showed clear photodynamic effects in both cell lines. The total fluence required to kill 50% of the cells at the drug concentration of 20 μM was 36 Jcm^-2 for HeLa cells and 72 Jcm^-2 for B16 cells. Neither PEG-based fullerene derivatives showed any appreciable photodynamic activity, possibly, due to low efficiency of singlet oxygen generation.

Biocompatible ZnS capped CdSe fluorescent semiconductor nanocrystals (quantum dots, QDs) exhibit great potential as imaging agents with biomedical and clinical relevance. However, little is known about the fate of the quantum dots in vivo, and the importance of chemical and physical composition that may influence their behavior in vivo. When the QDs are introduced in vivo, the first interactions with blood components will dictate their kinetic behavior in vivo. We present some preliminary results that demonstrate the interactions of the quantum dots with plasma proteins and that quantum dots can be trapped in fibrous networks.

We report on a new strategy for producing self-contained sensor elements for protein detection. The method exploits molecular imprinting, sol-gel-derived xerogels, and selective installation of the fluorescent reporter molecule within the template site. There are no biological reagents used. We term these new xerogel-based sensor elements as Protein Imprinted Xerogels with Integrated Emission Sites (PIXIES). The analytical figures of merit are described.

The templated growth of GaAs nanowires based on a catalytic seed particle is described. The optimum growth conditions for biosensor applications of the nanowires are presented including the catalyst formation process and nanowire growth temperature. We have also observed growth conditions that result in nanowires oriented parallel to the surface, which may result in a new paradigm for biosensor fabrication.

Immobilized gold nanoparticles on oxide surfaces were prepared by template assisted organometallic chemical vapor deposition (OMCVD). The growth parameters, optical parameters such as the plasmon resonance band, as well as geometrical parameters such as size and shape are investigated by UV-Vis spectroscopy, AFM, TEM and SEM. These new kind of gold nanoparticles were applied on a fluorescence based oligonucleotide hybridization study. Gold nanoparticles with a mean lateral diameter of 12 nm yielding an extinction maximum at around 520-530 nm should be able to resonantly excite the Cy3-fluorescence label attached to the target oligonucleotide. The hybridization reaction is taking place between gold nanoparticle surface-attached oligonucleotide catcher strands and chromophore-labeled target strands in solution. The experiment was conducted in the form of classical total internal reflection fluorescence detection. The kinetic data were quantitatively analyzed following a simple Langmuir model. It was found that a single mismatch between oligonucleotide target and probe reduces the Langmuir equilibrium constant by two orders of magnitude, allowing for an excellent sequence-specific detection of oligonucleotide hybridization based on OMCVD gold nanoparticles.

Polylysine and aminopropylsilane treated glass comprised the majority of substrates employed in first generation genetic microarray substrates. Second generation single stranded long oligo libraries with amino termini provided for controlled terminal specific attachment, and rationally designed unique sequence libraries with normalized melting temperatures. These libraries benefit from active covalent coupling surfaces such as Epoxysilane. The latter's oxime ring shows versatile reactivity with amino-, thiol- and hydroxyl- groups thus encompassing small molecule, oligo and proteomic microarray applications. Batch-to-batch production uniformity supports entry of the Epoxysilane process into clinical diagnostics. We carried out multiple print runs of 21 clinically relevant bacterial and viral antigens at optimized concentrations, plus human IgG and IgM standards in triplicate on multiple batches of Epoxysilane substrates. A set of 45 patient sera were assayed in a 35 minute protocol using 10 microliters per array in a capillary-fill format (15 minute serum incubation, wash, 15 minute incubation with Cy3-labeled anti-hIgG plus Dy647-labeled anti-hIgM, final wash). The LOD (3 SD above background) was better than 1 microgram/ml for IgG, and standard curves were regular and monotonically increasing over the range 0 to 1000 micrograms/ml. Ninety-five percent of the CVs for the standards were under 10%, and 90% percent of CVs for antigen responses were under 10% across all batches of Epoxysilane and print runs. In addition, where SDs are larger than expected, microarray images may be readily reviewed for quality control purposes and pin misprints quickly identified. In order to determine the influence of stirring on sensitivity and speed of the microarray assay, we printed 10 common ToRCH antigens (H. pylori, T. gondii, Rubella, Rubeola, C. trachomatis, Herpes 1 and 2, CMV, C. jejuni, and EBV) in Epoxysilane-activated slide-wells. Anti-IgG-Cy3 direct binding to printed IgG calibration spots could be detected (3 x LOD) above background at 100 pg/ml (0.13 femtomoles sample content) in a 10 minute incubation. The LOD for detection of serum anti-H. pylori antibody level was 9 ng/ml in the same incubation time.

Fluorescent reporter groups are widely used in many areas of macromolecular science. In nucleic acid chemistry, fluorescently labeled oligonucleotides find use in the study of nucleic acid conformations, as reporters of the state of hybridization and as probes of nucleic acid-protein interactions, as well as, probes and primers for the detection and quantification of desired sequences. Often, the fluorophore is a separate and independent entity covalently attached to an otherwise unmodified oligonucleotide. Another class of fluorophore is the nucleobase analog - a chemically modified nucleobase that is unable to base pair properly. Recently, we have become interested in pyrrolocytosine (pC) as an intrinsically fluorescent, pairing-competent nucleobase. It has previously been shown that incorporation of pyrrolocytidine into oligonucleotides gives a fluorescent molecule that responds to the state of hybridization. We have refined the syntheses of pyrrolocytosine derivatives and the related modified uracil derivatives, furanouracil (fU), from cytosine and uracil respectively via three sequential steps: iodination of the nucleobase; Pd-catalyzed cross-coupling with a terminal alkyne; and metal-catalyzed cyclization. The fluorescence properties of the 5-alkynyl nucleobases and the bicyclic pC and fU analogs have been characterized and depend on: 1) the nature of the substituent on the alkyne, 2) whether the nucleobase is 5-alkynyl or cyclized, and 3) the presence of a substituent on the N¹-position of the nucleobase. The chemistry and properties of peptide nucleic acid oligomers incorporating these types of base modifications and their possible use in point-mutation detection is presented.

Biosensors and biochips can determine the presence of nucleic acid sequences in a test sample through fluorescence detection of hybridization between an immobilized nucleic acid (probe) and a nucleic acid in a test sample (target). The finding that the control of the environment of immobilized single-stranded probe molecules on fused silica surfaces can be used to tune selectivity to facilitate detection of even single base pair mismatches provides opportunities for design of novel biochips. We have typically used silane coupling agents to activate silicon and silicate surfaces for subsequent immobilization of biomolecules for development of optical biosensors. A self-assembled immobilization providing good structural order would be preferred. Studies have been done using thiol-terminated reagents for assembly of oligonucleotides on GaAs substrates. The spacing of Ga and As can be controlled across a surface, and in turn provides a template to control the density of self-assembled oliogonucleotide. Initial experimental work has begun using homogeneous GaAs surfaces, and the homogeneity and surface morphology of immobilized oligonucleotide films grown onto GaAs has been characterized by atomic force microscopy (AFM) and fluorescence methods. Cycles of hybridization and denaturation suggest that the GaAs provides a surface that is stable to loss of immobilized oligonucleotide, but that efforts to protect from non-selective adsorption are essential. Data suggested that the films were of monolayer thickness, and that it was possible to induce the presence of nodules of approximately 10-50 nm in diameter.

We report the design and the synthesis of membrane-active peptide nanostructures, as well as their use as signal transducer in a fluorimetric assay for biologically relevant analytes. Addition of hydrophobic 21-residue peptides bearing six crown ether side chains to a solution of small unilamellar vesicles loaded with a pH-sensitive fluorophore induces a rapid fluorescence increase associated with Na⁺/H⁺ transport across the bilayer membrane. To demonstrate the usefulness of these peptide nanostructures in the development of simple, rapid, and sensitive detection assays for a wide range of analytes, peptide nanostructures bearing a biotin at the N-terminal position were prepared. Addition of avidin to the assay employing these modified peptides resulted in a significant change in the time-dependent fluorescence profile. Control experiments with a non-binding proteins and saturated avidin showed that the observed changes are indeed due to specific binding of avidin to biotin modified nanostructures.

Advances in DNA microarray fabrication technologies, expanding probe libraries, and new bioinformatics methods and resources have firmly established array-based techniques as mainstream bioanalytical tools and the application space is proliferating rapidly. However, the capability of these tools to yield truly quantitative information remains limited, primarily due to problems inherent to the use of fluorescence imaging for reading the hybridized arrays. The obvious advantages of fluorescence are the unrivaled sensitivity and simplicity of the instrumentation. There are disadvantages of this approach, however, such as difficulties in achieving optimal labeling of targets and reproducible signals (due to quenching, resonance energy transfer, photobleaching effects, etc.) that undermine precision. We are exploring alternative approaches, based mainly on Raman and resonance Raman spectroscopy, that in principle permit direct analysis of structural differences between hybridized and unhybridized probes, thereby eliminating the need for labeling the target analytes. We report here on the status of efforts to evaluate the potential of these methods based on a combination of measured data and simulated experiments involving short (12-mer) ssDNA oligomer probes with varying degrees of hybridized target DNA. Preliminary results suggest that it may be possible to determine the fraction of duplex probes within a single register on a DNA microarray from 100% down to 10% (or possibly less) with a precision of ±2 5%. Details of the methods used, their implementation, and their potential advantages and limitations are presented, along with discussion of the utility of using 2DCOS methods to emphasize small spectral changes sensitive to interstrand H bonding, backbone flexibility, hypochromicity due to base-stacking in duplex structures and solvation effects.

When monitoring separation events in microfluidic devices, one frequently needs to detect small amounts of analyte in picolitre sized volumes with a time response of milliseconds. Fluorescence detection is typically the method of choice due to its very high sensitivity and fast response. However, since many analytes are not naturally fluorescent, labelling protocols may have to be introduced and thereby increase the complexity of the analysis. Here, we present an alternative method that is based on optical absorption, or more specifically on the ring-down time of an optical signal in a cavity or loop made of waveguide material. This optical decay constant changes as small liquid samples containing absorbing species are introduced into a fiber-optic loop. It is demonstrated that one can obtain the optical decay constant using a continuous wave laser beam that is intensity modulated and then coupled into an optical fiber loop. The inherent exponential decay in the fiber loop introduces a phase shift of the light emitted from the loop with respect to the pumping beam. By measuring this phase shift, one can readily determine the concentration of the analyte introduced between the two fiber ends and a model is established to describe the relationship. It is demonstrated that this technique, dubbed "phase-shift fiber-loop ring-down spectroscopy" (PS-FLRDS), is well suited as an absorption detector for any flow system in which the optical absorption path is limited by the instrument architecture.

Ultrasensitive optical absorption detection method based on multi-photon wave-mixing spectroscopy is inherently suitable for biochips, biosensors and microarrays. Absorption-based wave mixing offers detection sensitivity levels that are comparable or better than those of fluorescence-based methods while using micrometer-thin samples. Wave-mixing methods offer detection of biospecific interactions on antibody or oligonucleotide microarray platforms. Optical absorption measurements of protein and DNA profiles on a glass substrate can be made with excellent reproducibility. The laser probe volume, i.e., the overlap volume of two input laser beams, ranges from nanoliter to picoliter levels. Dynamic gratings created inside the absorbing sample diffract off incoming photons to create the signal beam. Unlike fluorescence methods, wave mixing generates a collimated laser-like signal beam, and hence, signal collection is efficient and convenient. Each microarray spot has a specific antibody or oligonucleotide sequence and wave mixing allows detection of corresponding target molecules with high throughput. Wave mixing also yields intra-spot spatial resolution for profiling individual array spots.

We report on development of frequency-doubled diode-pumped ultrashort pulse Yb:KGW laser operating at 520 nm with approximately 200 fs long pulses at a repetition rate of 15 MHz. For ~2 W of absorbed pump power at 980 nm, the laser delivers up to 30 mW of average power at fundamental wavelength of 1040 nm, corresponding to a pulse energy of 2 nJ. The laser radiation was then frequency-doubled in a single pass configuration within a nonlinear BIBO crystal to produce femtosecond green radiation at 520 nm with peak power of ~200 W. The generated second harmonic served as excitation source of optical DNA sensor based on fluorescence lifetime measurements using the time correlated single photon counting (TCSPC) technique.

The results of preliminary investigations toward the design of an optical biosensor instrument for the selective and direct analysis of low copy numbers of target nucleic acids in native form are reported. A concept development prototype was constructed based on a total internal reflection fluorescence (TIRF) configuration and the use of time correlated single photon counting (TCSPC). Selective detection of interfacial hybrid formation was done by identification of luminescence of characteristic (20ns) lifetime from the intercalant fluorophore ethidium bromide associated with nucleic acid hybrids formed at the interaction surface of optical sensor elements. Results of these investigations suggest that detection limits on the order of 10⁷ dye:dsDNA complexes can be achieved when an effective sensor interaction surface of 150 µm diameter is used. The presence of interfacial nucleic acid duplexes at a sensor surface was further verified by thermal denaturation studies. The sensitivity of this concept design prototype was found to be most limited by long lifetime fluorescence intrinsic to the detection optics in conjunction with large amounts of scatter dispersed from the sensor cartridge. Future directions for continued device development are discussed.

Fiber-optic waveguides based Micro-Opto-Electro-Mechanical Systems (MOEMS) form a significant class of biosensors which have notable advantages like light weight, low cost and more importantly, the ability to be integrated with bio-systems. In this work, integrated microfluidic fiber-optic waveguide biosensor is presented. The phenomenon of evanescence is employed for sensing mechanism of the device. Herein, the fiber-optic waveguide is integrated with bulk micromachined fluidic channel across which different chemical and biological samples are passed through. The significant refractive index change due to the presence of biological samples that causes the evanescent field condition in the waveguides leads to optical intensity attenuation of the transmitted light. The study of the modulation in optical intensity is used to detect the properties of the species used in the evanescent region. The intensity modulation of light depends upon the geometry of the waveguide, the length of evanescent field, the optical properties of specimen used for producing evanescence and the changes in the properties by their reaction with other specimen. Therefore, this device is proposed for biosensing applications. The Finite Element Analysis (FEA) has been carried out for wave propagation under the evanescent condition for different parametric variations.

Miniaturization and highly accurate detection technologies are key factors to advancing sensor performance and utility and the search for such technologies promises accomplishments with the advent of Micro Electro Mechanical Systems (MEMS) structures. MEMS is an enabling technology that can create integrated devices with mechanical, optical and electronic components. One of the fields where these micro-devices are successfully widespread is that of medical care where micro-machined cantilever sensors are found to be the ideal candidates for bio-sensing applications. These micro-machined cantilevers have been proposed as mechanical transducers for different sensing applications. These sensing surfaces are of interest in the development of novel cantilever-based biosensors. The fascinating aspect about these transducers is that they bend due to modifications in nano-mechanical interactions between neighboring molecules which curve the beam and that curvature can be optically detected. In this paper, PVDF-cantilevers are coated with 2 sets of antibody and antigen on one side, which respond with specific deflection signatures to each other and their intermolecular nano-mechanics bend the cantilever. The first set of antibody and antigen used here are rabbit skeletal muscle Troponin C (TnC) and Honey Bee Venom Melittin (ME) prepared in 50mM KCl and 50mM Tris-HCl buffer at a pH of 7.5 in a 1:1 ratio. The next set used were Horse Raddish Peroxide (HRP) and Hydrogen Peroxide (H2O2) prepared to get 10mg per 1ml of 0.1M Potassium Phosphate dibasic (K2HPO4) and diluted hydrochloric acid to get pH of 6.0. The optical system includes a laser source and a Position Sensitive Detector (PSD) which is used to readout deflections of the PVDF-cantilevers. The behavior of the cantilevers was also monitored with the enzymes under the influence of voltage. The classical problem of evaluating the tip deflection of the cantilever beam is analyzed involving the relation between movement recorded by the PSD, the tip deflection and angle of incidence. The combined results provide valuable information on the development of an optimizing sensing element that would enable life saving treatments of patients suffering from Acute Myocardial Infarction (AMI).

Single molecule fluorescence techniques have contributed considerably to our understanding of a variety of biological systems. Unfortunately, single molecule techniques are fundamentally limited by the concentration of fluorescent species and volume being observed. Nanofabricated structures have emerged as ideal tools for volume confinement in fluorescence spectroscopy. This has allowed the extension of single molecule techniques to high concentration regimes. Zero Mode Waveguides, nanometer scale holes in a thin metal film produce observation volumes in the attoliter to zeptoliter range. These structures have been used to measure protein kinetics in the micro molar concentration range as well as for observations of polymerase incorporating single nucleotide bases. We have investigated the optical properties of these structures using a combination of fluorescence spectroscopy, scanning electron microscopy and optical transmission measurements in an effort to accurately model the optical properties of these structures. These measurements have allowed a detailed characterization of the optical behavior of sub-wavelength apertures in a thin metal film.

In recent years, capillary electrophoresis (CE) has been one of rapidly growing analytical techniques to study affinity interactions. Quick analysis, high efficiency, high resolving power, low sample consumption, and wide range of possible analytes make CE an indispensable tool for studies of biomolecules and, in particular, studies of their interactions. In the article, we discuss kinetic methods in CE. The spectrum of proven applications of kinetic CE methods includes: (i) measuring equilibrium and rate constants of protein-ligand interaction from a single experiment, (ii) quantitative affinity analyses of proteins, (iii) measuring temperature in CE, (iv) studying thermochemistry of affinity interactions, and (v) kinetic selection of ligands from combinatorial libraries. We demonstrate that new kinetic CE method can serve as a "Swiss army knife" in the development and utilization of oligonucleotide aptamers. Uniquely, they can facilitate selection of smart aptamers - aptamers with pre-defined binding parameters. We believe that further development of kinetic CE methods will provide a variety of methodological schemes for high-throughput screening of combinatorial libraries for affinity probes and drug candidates using CE as a universal instrumental platform.

Two possible system integration approaches for portable real time cytometry in microfluidic applications are discussed. An ocular mounted linear CMOS image sensor configured for real time detection of particles being transported in microfluidic channels is first described. This system delivers cytometry functionality utilizing standard microfluidic chips and conventional optics. While this approach affords a certain ease of integration, one significant drawback is the singular field of view onto the microfluidic substrate and the reliance on conventional microscopy. However, the microscopy resolution makes possible a simple device capable of determining precise position, size and trajectory information on a per particle basis. A second architecture comprises flip-chip integration of a custom CMOS active pixel sensor aboard a custom microfluidic glass substrate. At the expense of optical resolution, the near field sensor topology obviates the need for conventional microscopy, affords simultaneous multi-channel sensing and takes strides towards cost effective micro total analysis system (uTAS) deployment. The two platforms share a common microcontroller for processing, control and display as well as a unified host-side application programming interface; an approach which will enable side-by-side comparison of the two hardware architectures in real time. The common user interface, a platform independent .NET application, deploys to both desktop and compact framework pocket PC platforms.

Microarray analysis has become increasingly complex due to the growing size of arrays. In this work we explore the effects of diffusion and convective fluxes on the time of hybridization and sensing specificity in a single component system. A .nite element software is used to simulate the diffusion of DNA through a microfluidic chamber to the sensing surface where hybridization of DNA is modeled using the corresponding kinetic equation. The differences between diffusion controlled and convection controlled mass transport are investigated as a function of concentration and hybridization time. Hybridization enhancement produced by microfluidics versus stationary diffusion is introduced as a useful metrics for quantitation of mass transport effects.

We present an optical microsystem aimed to be integrated into a nanomechanical biosensor for functional genomic analysis. The operation principle is based on a sub-nanometer resolution optical measurement of a cantilever deflection caused by a surface stress when the target nucleic acid sample hybridises to the nucleic acid probe on the active side of the cantilever. The resulting deflection, of the order of nanometers, is measured by an optical system, in which a laser beam reflects off the back of the cantilever to a position sensitive photo-detector. We report in this paper on the design, fabrication and test of the optical head associated with an optical coupling system which enables detection of the presence of target nucleic acid on the cantilever by amplifying the deflection caused by the stress.

The bactericidal effect of visible light illumination on bacteria treated with non-toxic photosensitizers (PS) has been shown previously, its effectiveness depended on both cell type and nature of photosensitizer used. The photosensitizer (PS)-mediated bactericidal effect of light against different types of microorganisms including vegetative bacteria (both in planktonic form and in biofilms), bacterial spores, yeasts, viruses was investigated for both cells in liquid media, and on surface. Bactericidal effect was monitored for different photosensitizer such as TBO and derivatives of rosamine at different concentrations. The possibility of using photodynamic treatment for surface sanitation was investigated.

Photodynamic therapy (PDT) based on simultaneous two-photon (2-γ) excitation has a potential advantage of highly targeted treatment by means of nonlinear localized photosensitizer excitation. One of the possible applications of 2-γ PDT is a treatment of exodus age-related macular degeneration where highly targeted excitation of photosensitizer in neovasculature is vital for reducing collateral damage to healthy surrounding tissue. To investigate effect of 2-γ PDT Photofrin was used as an archetypal photosensitizer. First, 2-γ absorption properties of Photofrin in the 750 - 900 nm excitation wavelength range were investigated. It was shown that above 800 nm 2-γ interaction was dominant mode of excitation. The 2-γ cross section of Photofrin was rather small and varied between 5 and 10 GM (1 GM = 10^-50 cm⁴s/photon) in this wavelength range. Next, endothelial cells treated with Photofrin were used to model initial effect of 2-γ PDT on neovasculature. Ultrashort laser pulses provided by mode-locked Ti:sapphire laser (pulse duration at the sample 300 fs, repetition rate 90 MHz, mean laser power 10 mW, excitation wavelength 850 nm) were used for the excitation of the photosensitizer. Before 2-γ excitation of the Photofrin cells formed a single continuous sheet at the bottom of the well. The tightly focused laser light was scanned repeatedly over the cell layer. After irradiation the cell layer of the control cells stayed intact while cells treated with photofrin became clearly disrupted. The light doses required were high (6300 Jcm(-2) for ~ 50% killing), but 2-γ cytotoxicity was unequivocally demonstrated.

This study investigates the use of photodynamic therapy (PDT) in regulating bone development with a view to its potential role in treating Juvenile leg length discrepancy (LLD). Transgenic mice expressing the luciferase firefly gene upon activation of a promoter sequence specific to the vascular endothelial growth factor (VEGF) gene were subject to benzoporphyrin derivative monoacid (BPD-MA)-mediated PDT in the right, tibial epiphyseal growth plate at the age of 3 weeks. BPD-MA was administered intracardially (2mg/kg) followed 10 mins later by a laser light (690 +/- 5 nm) at a range of doses (5-27J, 50 mW output) delivered either as a single or repeat regimen (x2-3). Contra-lateral legs served as no-light controls. Further controls included animals that received light treatment in the absence of photosensitizer or no treatment. Mice were imaged for VEGF related bioluminescence (photons/sec/steradian) at t= 0, 24, 48, 72 h and 1-4 weeks post PDT. Faxitron^TM x-ray images provided accurate assessment of bone morphometry. Upon sacrifice, the tibia and femur of the treated and untreated limbs were harvested, imaged and measured again and prepared for histology. A number of animals were sacrificed at 24 h post PDT to allow immunohistochemical staining for CD31, VEGF and hypoxia-inducible factor (HIF-1 alpha) within the bone. PDT-treated (10 J, x2) mice displayed enhanced bioluminescence at the treatment site (and ear nick) for up to 4 weeks post treatment while control mice were bioluminescent at the ear-nick site only. Repeat regimens provided greater shortening of the limb than the corresponding single treatment. PDT-treated limbs were shorter by 3-4 mm on average as compared to the contra lateral and light only controls (10 J, x2). Immunohistochemistry confirmed the enhanced expression VEGF and CD31 at 4 weeks post-treatment although no increase in HIF-1α was evident at either 24 h or 4 weeks post PDT treatment. Results confirm the utility of PDT to provide localized effects on bone development that may be applicable to other related skeletal deformities.

Photodynamic therapy (PDT) as a non-radiative treatment has been applied successfully in various cancers. PDT may be a useful adjunct in the treatment of vertebral metastases. PDT efficacy requires the administration of a photosensitiser drug followed by subsequent drug activation by wavelength specific light. The study purpose was to establish the pharmacokinetic profiles for 2 photosensitisers, BPD-MA and 5-ALA induced PpIX, to determine the optimal drug-light interval for vertebral PDT.

The work is devoted to photoluminescent investigation of arterial walls in order to create a new navigation method for minimally invasive treatment of cardiovascular decease in the presence of chronic total occlusions. The method uses the distinct photoluminescent properties of arterial wall and chronic total occlusion plaque to alert the interventionalist when a fiber-optic equipped catheter is in contact with the vessel wall. We conducted a study to compare the photoluminescence properties of healthy and stenosed vessel walls, and a typical chronic total occlusion plaque in the spectral range 300-700 nm. All samples were obtained from human tibial arteries. These groups of arterial samples showed easily differentiable luminescence amplitude and spectral characteristics. The photoluminescent properties of intact and intentionally damaged vessel walls were also investigated to permit detection of artery perforation that could take place during the revascularization. Using optical excitation of different wavelength gives additional opportunities of detecting arterial plaques requiring laser treatment. The results presented are complemented with micro-computed tomography images and histology of the segments analyzed.

We discuss image reconstruction algorithms for diffuse optical tomography that allow utilization of extremely large data sets. Image reconstruction is performed with experimental data obtained with the use of a CCD camera-based noncontact imager. We demonstrate that more than 10⁷ measurements can be acquired and utilized. This is two orders of magintude or more larger than the data sets which are typically used in diffuse optical imaging.

Optical aberrations reduce the imaging quality of the human eye. In addition to degrading vision, this limits our ability to illuminate small points of the retina for therapeutic, surgical or diagnostic purposes. When viewing the rear of the eye, aberrations cause structures in the fundus to appear blurred, limiting the resolution of ophthalmoscopes (diagnostic instruments used to image the eye). Adaptive optics, such as deformable mirrors may be used to compensate for aberrations, allowing the eye to work as a diffraction-limited optical element. Unfortunately, this type of correction has not been widely available for ophthalmic applications because of the expense and technical limitations of current deformable mirrors. We present preliminary design and characterisation of a deformable mirror suitable for ophthalmology. In this ferrofluidic mirror, wavefronts are reflected from a fluid whose surface shape is controlled by a magnetic field. Challenges in design are outlined, as are advantages over traditional deformable mirrors.

Many diagnostic and therapeutic approaches in medical physics today take advantage of the unique properties of light and its interaction with tissues. Because light scatters in tissue, our ability to develop these techniques depends critically on our knowledge of the distribution of light in tissue. Solutions to the diffusion equation can provide such information, but often lack the flexibility required for more general problems that involve, for instance, inhomogeneous optical properties, light polarization, arbitrary three-dimensional geometries, or arbitrary scattering. Monte Carlo techniques, which statistically sample the light distribution in tissue, offer a better alternative to analytical models. First, we discuss our implementation of a validated three-dimensional polarization-sensitive Monte Carlo algorithm and demonstrate its generality with respect to the geometry and scattering models it can treat. Second, we apply our model to bioluminescence tomography. After appropriate genetic modifications to cell lines, bioluminescence can be used as an indicator of cell activity, and is often used to study tumour growth and treatment in animal models. However, the amount of light escaping the animal is strongly dependent on the position and size of the tumour. Using forward models and structural data from magnetic resonance imaging, we show how the models can help to determine the location and size of tumour made of bioluminescent cancer cells in the brain of a mouse.

The work that is presented in this paper proposes a new low-cost and compact optoelectronic system for optical mammography trials. This system is based on the use of cost-off-the-shelf (COTS) optoelectronic and electronic components which are commonly used in the telecommunications industry. The main components of this novel system are based on low cost semiconductor laser diodes and avalanche photodiodes (APD's). This is backed up by the use of a versatile electronic architecture also based on telecommunication low cost components and techniques. Such methods allow research into the optimum modulation frequency for improved signal to noise ratio (SNR). To date the system has been tested on liquid phantoms formed by different concentrations of milk solutions. Results obtained from preliminary tests are within 10% of values obtained in previous publications.

Alzheimer's disease currently affects over 800,000 people in the UK, and this figure is set to exceed 1.5 million by 2050. The disease causes a severe breakdown of the brain, resulting in the progressive decline in the mental health of the patient. It also remains without a long-term cure. A definitive diagnosis of the disease can only be gained at post-mortem, whilst other technologies are limited to monitoring the physical breakdown of the brain. The early identification of the chemicals responsible for the disease, and their structure, is therefore essential for improved diagnosis, treatment and care. This research demonstrates the use of Raman spectroscopy, and principle components analysis, as a method for the diagnosis, and examination of, the specific proteins responsible for Alzheimer's disease. Analyses of ethically approved ex vivo brain tissues from normal elderly (n=3), Alzheimer's disease (n=12) and Huntingdon's disease (n=3) brain sections (from the frontal and occipital lobes) are presented. Subsequently, a further 12 blinded individual samples were examined and the preliminary results are presented. Spectra originating from these tissues are highly reproducible, and results indicate a vital difference in protein content and protein conformation, relating to the abnormally high levels of aggregated proteins in the diseased tissues. Principle components analysis has been shown to differentiate normal elderly tissues from diseased tissues. These results show great promise for the early identification of Alzheimer's disease, and secondary information regarding other brain diseases and dementias. These results demonstrate the potential use of Raman spectroscopy as a possible non-invasive, non-destructive tool for the early diagnosis of Alzheimer's disease.

Along with breast and cervical cancer, esophageal adenocarcinoma is one of the most common types of cancers. The characteristic features of pre-cancerous tissues are the increase in cell proliferation rate and cell nuclei enlargement, which both take place in the epithelium of human body surfaces. However, in the early stages of cancer these changes are very small and difficult to detect, even for expert pathologists. The aim of our research is to develop an optical probe for in vivo detection of nuclear size changes using white light scattering from cell nuclei. The probe will be employed through an endoscope and will be used for the medical examination of the esophagus. The proposed method of examination will be noninvasive, cheap, and specific, compared to a biopsy. Before the construction of this probe, we have developed theory to determine the nuclei size from the reflection data. In this first stage of our research, we compare experimental and theoretical scattered light intensities. Our theoretical model includes the values of scatterer size from which we can extract the nuclei size value. We first performed the study of polystyrene microspheres, acting as a tissue phantom. Spectral and angular distributions of scattered white light from tissue phantoms were studied. Experimental results show significant differences between the spectra of microspheres of different sizes and demonstrate almost linear relation between the number of spectral oscillations and the size of microspheres. Best results were achieved when the scattered light spectrum was collected at 30° to the normal of the sample surface. We present these research results in this paper. In ongoing work, normal and cancerous mammalian cell studies are being performed in order to determine cell nuclei size correlation with the size of microspheres through the light scattering spectrum observation.

This study aimed at applying Laser induced-autofluorescence (LIAF) diagnostics method as an in-vivo screening of colorectal polyplcancer. The spectrum algorithm based on the ratio of autofluorescence intensity was used to identify the diseased tissues from the normal tissues as it was generally performed better than an algorithm based only simply on the intensity of the spectrum. Histopathological biopsy results were compared with the detected AF spectra characteristics for different kinds of polyps. 73 patients had been examined via the LIAF spectroscopy detection system during their colonoscopy screening in Endoscopy Center, Singapore General Hospital. The autofluorescence from the surface of the colorectal tissues under 405 nm laser light excitation was detected using our detecting system. In the experimental investigation two groups of patients were involved. One group was "abnormal" group. There were 25 patients belonging to this group since polyps or carcinoma was found in their colorectal tract during colonoscopy. The histopathology reports confirm the group classification. Total 36 polyps' AF spectra and 9 carcinoma' AF spectra were detected from 25 patients of the abnormal group during their regular endoscopy examination. The intensity ratios R_I-680/I-500 and R_I-630/I-500 of polyps/cancerous AF spectra and intensity ratios of corresponding normal colorectal AF spectra were calculated. Two critical intensity ratios for separating the AF intensity ratios R_I-680/I-500 and R_I-630/I-500 of normal and abnormal colorectal tissues were defined as 0.5 and 0.6 respectively. Using the critical intensity ratio values, 48 "normal" group patients' rectums were checked via the LIAF detection system. There were 20 patients (41.7%) whose AF spectra of colorectal tract mucosa belonging to abnormal spectra. However, these 20 patients had not been found under white light via traditional endoscopy. For small diseased area like small plat polyp disease and carcinoma, it was very difficult to identify under white light by endoscopy. However, the LIAF spectra technique and AF intensity ratio algorithm was able to detect these kinds of abnormal area earlier than traditional endoscopy. Using this algorithm, it is able to identify the onset of abnormal tissue growth during real-time clinical endoscope examination.

Pressure ulcers (sores) can occur when there is constant pressure being applied to tissue for extended periods of time. Immobile people are particularly prone to this problem. Ideally, pressure damage is detected at an early stage, pressure relief is applied and the pressure ulcer is averted. One of the hallmarks of pressure damaged skin is an obliterated blanch response due to compromised microcirculation near the surface of the skin. Visible reflectance spectroscopy can noninvasively probe the blood circulation of the upper layers of skin by measuring the electronic transitions arising from hemoglobin, the primary oxygen carrying protein in blood. A spectroscopic test was developed on a mixed population of 30 subjects to determine if the blanch response could be detected in healthy skin with high sensitivity and specificity regardless of the pigmentation of the skin. Our results suggest that a spectroscopic based blanch response test can accurately detect the blanching of healthy tissue and has the potential to be developed into a screening test for early stage I pressure ulcers.

The effects of optically turbid medium on polarization states of incident light are studied using a novel linear Stokes polarimeter. The optical rotation and surviving linear polarization fraction of light scattered from highly turbid media (scattering coefficient μ_s = 100 cm^-1) are measured both in and off the incident plane while the detection angle changes from forward direction (0°) to backward directions (135°, 145° and 155°). The response of the optical rotation and surviving linear polarization to the presence of glucose molecules (0.06M - 0.9M) is also studied. The results show that in the absence of glucose, the scattering-induced optical rotation is zero in the incident plane for all detection angles, and increases with detection angle when measured off the incident plane. Conversely, the surviving linear polarization fraction increases with detection angle in the incident plane, and decreases when off the incident plane. Thus, when measured in the incident plane, optical rotation is least sensitive to glucose in the turbid medium, whereas the surviving linear polarization is most sensitive. For the above turbidity and glucose concentration ranges, the optimal glucose detection sensitivity using optical rotation is at 135° detection angle, 2 mm off the incident plane, while it is at 135° detection angle in the incident plane if surviving linear polarization is used as a glucose probe. This work demonstrates the complexity of polarimetry in turbid chiral media and underscores the importance of detection geometry in making and interpreting turbid polarimetry measurements.

Tissue undergoing transformation into a state that is more favourable for tumor growth may present itself with different tissue optical properties and contain different amounts of the major tissue chromophores. Here, we decomposed transillumination spectra obtain in women from various risk levels of developing breast cancer.

Medical diagnostics and screening are becoming increasingly demanding applications for spectroscopy. Although for many years the demand was satisfied with traditional spectrometers, analysis of complex biological samples has created a need for instruments capable of detecting small differences between samples. One such application is the measurement of absorbance of broad spectrum illumination by breast tissue, in order to quantify the breast tissue density. Studies have shown that breast cancer risk is closely associated with the measurement of radiographic breast density measurement. Using signal attenuation in transillumination spectroscopy in the 550-1100nm spectral range to measure breast density, has the potential to reduce the frequency of ionizing radiation, or making the test accessible to younger women; lower the cost and make the procedure more comfortable for the patient. In order to determine breast density, small spectral variances over a total attenuation of up to 8 OD have to be detected with the spectrophotometer. For this, a high performance system has been developed. The system uses Volume Phase Holographic (VPH) transmission grating, a 2D detector array for simultaneous registration of the whole spectrum with high signal to noise ratio, dedicated optical system specifically optimized for spectroscopic applications and many other improvements. The signal to noise ratio exceeding 50,000 for a single data acquisition eliminates the need for nitrogen cooled detectors and provides sufficient information to predict breast tissue density. Current studies employing transillumination breast spectroscopy (TIBS) relating to breast cancer risk assessment and monitoring are described.

Near-infrared (NIR) spectroscopy is being applied to the solution of problems in many areas of biomedical and pharmaceutical research. The need for modern medical diagnostics to develop small portable instruments that enable fast and effective monitoring of the biological properties of the human body is apparent. We have developed a portable and robust spectrometer that consists of a two beam interferometer operating in the near-infrared wavelength range for real-time measurements. The device has limited spectral resolution and so methods of computational intelligence and advanced signal processing have been applied to the NIR data to produce more precise and informative diagnostic information. Our target application concerns blood and tissue status in a form that can be interpreted directly by the user, without special knowledge of spectral analysis. More specifically, theories and methods from the field of machine intelligence (learning algorithms, neural networks, etc.) were first applied to classify in vitro urea samples of different concentrations. The results are encouraging, with overall mean squared prediction errors of less than 10^-4, and in vivo trials will follow to further develop the device. Non-intrusive diagnostics of this kind are suitable for point-of-care screening.

The process of taking a concept to a clinical device begins with the idea for a technological solution to an unmet clinical challenge. Burns are one of the most destructive insults to the skin causing damage, scarring, and in some cases death. The approach most commonly used to evaluate burns is based on the appearance of the wound. This technique is somewhat subjective and unreliable, relying on clinical experience to assess the burn. Instrument based diagnostic techniques as an adjunct to current practices has the potential to enhance the quality and timeliness of decisions concerning wound assessment and treatment. Near Infrared Spectroscopy is a promising technique that can track changes within the tissue, and can therefore provide insight as to how deep the burn actually penetrates before visual signs become apparent. Preliminary bench and animal studies were used to prove the concept of a near infrared based method of burn assessment. This study demonstrated the ability of near infrared imaging to detect and monitor the hemodynamics of burn injuries in the early post-burn period. Based on this study, a pre-prototype near infrared spectroscopic system was built with the goal of developing a reliable yet simple system that could be used in a clinical setting. A pilot clinical study was designed and implemented at the Ross Tilley Burn Center (Toronto, Canada) in order to assess the feasibility of our strategy in the clinical realm. The goal of this preliminary clinical study was to determine if the pre-prototype could be integrated into the strict regiment of an active burn centre. Both the instrument performance in a clinical setting and the injury assessment based on the analysis of near infrared reflectance measurements were a success.

Infrared spectroscopy is well established as an analytical technique in various applications. We have undertaken a series of studies to establish the suitability of mid infrared spectroscopy in various clinical analytical applications, focusing on various urine, serum and whole blood assays. The initial work demonstrated that six common serum analyses are possible, namely glucose, urea, total cholesterol, triglycerides, total protein, and albumin, with accuracy comparable to standard clinical methods (Hitachi 717), and more recently HDL and LDL cholesterol have been quantified separately. Herein, we summarize our progress in transferring this technology to the clinical laboratory, focusing on the new methods and hardware that have enabled this transition, assessing the accuracy of the mid IR based analytical methods using these innovations, and reporting an exploratory study assessing the transferability of methods between spectrometers.

A spatially-multiplexed swept-source optical coherence tomography (SM-SS-OCT) system for rapid acquisition of B-scans of tissue microstructure is described, we believe, for the first time. SM-SS-OCT instrumentation is similar to that of traditional Swept Source OCT (SS-OCT), which uses a widely tunable (~100 nm) laser source to obtain high-resolution images of biological tissue. However, SM-SS- OCT may be considered an improvement over SS-OCT in terms of efficient usage of the wide spectral bandwidth afforded by the frequency-tunable lasers in SS-OCT systems. Commercially available swept-source lasers regularly achieve extremely narrow line widths (~150 KHz), allowing for SS-OCT A-scan depths on the order of meters. Since imaging tissue to such depths is infeasible, the meters-long depth ranging capability of SS-OCT may be utilized for spatially multiplexing many A-scans, each to lesser depth. We achieve this spatial multiplexing by rapidly scanning all lateral positions of the tissue repetitively while simultaneously scanning the laser wavelength continuously, and using appropriate signal processing to reconstruct a B-scan image from acquired data. Our fiber-based design lends itself towards use in endoscopic applications, and our results suggest that SM-SS-OCT can provide rapid acquisition of B-scans, with potential for depth-resolved visualization of transient processes in biological tissue.

Time domain Doppler optical coherence tomography (DOCT) is a promising non-invasive imaging system with high spatial (~20μm) and velocity resolution (~20μm/s) that can image microvascular blood flow. It is important to understand and account for the complicated 3D nature of small blood vessels. To address this problem, two realistic flow phantoms were designed with known geometries -- an occluded flow path to model vessel narrowing, and a Y-bifurcation to simulate vessel branching. The current DOCT system produces 2D images, which when stacked sequentially can yield 3D images of microstructure and perfusion-level blood flow. 3D reconstructions allow the investigation of internal flow profiles, including an abrupt stenosis in the occluded phantom. This research will help guide our image interpretation of in-vivo DOCT studies, including treatment response monitoring in animal tumours and endoscopic assessment of the human GI tract.

Chronic total occlusions (CTOs) are defined as complete occlusions of an artery older than one month. Minimally invasive catheter-based interventions commonly employed for partial occlusions (e.g., balloon angioplasty followed by stenting) are problematic in CTOs because of the phycisian's inability to pass the device through the occlusion without a significant risk of arterial wall perforations. Furthermore, successfully treated CTOs exhibit a high re-occlusion rate. As a result, these cases are mostly sent to bypass surgery. With the advent of drug-eluting stents that reduce the incidence of re-occlusion, and thus, eliminating the second problem, new devices have begun to emerge that aim to recanalize CTOs without the cost and trauma of bypass surgery. These devices, however, need effective image guidance methods to ensure successful crossing of the CTOs. Optical coherence tomography (OCT) is being evaluated as an intravascular imaging modality for guiding catheter-based interventions of CTOs. Occluded ex vivo human arterial samples were used to produce longitudinal cross-sections using an OCT system. These OCT images were compared with histology to assess OCT's ability to identify different components of the occluded artery, evaluate the imaging depth, and determine the ability to detect the underlying vessel wall. Given the inherent difficulties of creating a mechanically scanning OCT probe in the distal tip of a catheter for use in a stenotic artery, we directed our initial efforts towards developing a "motionless" fiber based OCT system using a single mode fiber array. We discuss design considerations for implementing a forward viewing intravascular OCT probe.

To design and deliver proper radiation treatment for cancer patients, knowledge of the body's surface in the affected area is required. Currently, surface information is obtained by using a manually operated tracer. The drawbacks of this contact method include slow operation, and errors in repositioning the patient in an x-ray machine. Utilization of MRI or CT is also possible but expensive. We propose a non-contact, quick, inexpensive method to reconstruct the surface. In our non-contact method, a mask with transparent circular coloured spots and a black background, and an incoherent light source are used to create structured-light images. Colour coding is necessary to establish the correspondence between the projected and the observed patterns, which is essential for surface reconstruction. The deformed light pattern is photographed by an offset camera and analyzed. First, noise reduction is performed because images are noisy due to the low-light conditions and low sensitivity of an off-the-shelf camera. Then, pattern elements (light elliptical spots) are found in the image. We use an inverse polynomial to model the intensity of a light spot, which results in a non-convex, least-squares optimization problem. Next, spots are assigned to a grid according to their colours and location, and errors are corrected using the relative position of the spots. Finally, spatial coordinates of the surface points are computed and surface reconstruction is performed. The described algorithms are implemented as a MATLAB package, which converts the acquired images into a three-dimensional surface. The developed system is inexpensive, and it can easily be mounted on an x-ray machine. The software package can run on any standard PC.

Atrial fibrillation (AF) is a heart rhythm abnormality that involves irregular, and often rapid, heartbeats. Recent studies demonstrate the feasibility of treating AF and other structural heart diseases with limited, left-atrial ablation lesion sets. These cardiac ablation procedures reduce the time required to perform the maze procedure surgery, and are less invasive. To produce long continuous transmural lesions, solid-state lasers and high power laser diodes, along with end emitting fiber optic catheters, have been used experimentally. These devices demonstrated promising results, but the absence of side emitting fiber flexible catheters to produce long continuous lesions limits the further development of this technology. In this research, a prototype energy delivery and control system located in a catheter, was demonstrated. The highlight of the proposed system is a flexible 10-cm fiber diffuser that can be used to make continuous photocoagulation lesions for effective maze procedure treatments. The system also includes: a flexible optical reflector; a distributed temperature sensor array for monitoring the temperature in the surrounding tissue; a series of openings for rapid self-attachment to the tissue (vacuum holder - gripper); and an optional closed-loop irrigating chamber with circulating coolant to cool the optical diffuser.

This study investigates the efficacy of low level laser therapy (LLLT) in modulating inducible nitric oxide synthase (iNOS) expression as molecular marker of the inflammation signaling pathway. LLLT was mediated by different therapeutic wavelengths using transgenic animals with the luciferase gene under control of the iNOS gene expression. Inflammation in 30 transgenic mice (iNOS-luc mice, from FVB strain) was induced by intra-articular injection of Zymosan-A in both knee joints. Four experimental groups were treated with one of four different wavelengths (λ=635, 785, 808 and 905nm) and one not laser-irradiated control group. Laser treatment (25 mW cm^-2, 5 J cm^-2) was applied to the knees 15 minutes after inflammation induction. Measurements of iNOS expression were performed at multiple times (0, 3, 5, 7, 9 and 24h) post-LLLT by measuring the bioluminescence signal using a highly sensitive charge-coupled device (CCD) camera. The responsivity of BLI was sufficient to demonstrate a significant increase in bioluminescence signals after laser irradiation of 635nm when compared to non-irradiated animals and the other LLLT treated groups, showing the wavelength-dependence of LLLT on iNOS expression during the acute inflammatory process.

Ultrashort laser pulses are very promising tools for performing accurate dissection in the eye, especially in the corneal stroma. The development of eye femtosurgery requires basic knowledge about laser-tissue interaction. One of the most significant parameters is the ablation threshold, the minimal laser energy per unit surface required for ablation. We present here measurements of the femtosecond laser ablation threshold as a function of the pulse duration for two cornea layers (epithelium and stroma) using optical damage diagnosis. Experiments have been realized with the INRS Ti:Sapphire laser (60 fs-5000 fs, 800 nm, 10 Hz). Our experimental results are fitted with a model for laser-matter interaction in order to determine some intrinsic physical parameters

UV irradiation is widely used in phototherapy. Regardless of the fact that UV overexposure is liable to cause adverse health effect, in appropriate doses UV radiation initiates synthesis of vitamin D in skin that is absolutely essential for human health. As it proved, most people in northern industrial countries have a level of vitamin D in their bodies that is insufficient for optimum health, especially in winter. These low levels of vitamin D are now known to be associated with a wide spectrum of serious disease much of which leads on to premature death. The diseases associated with D deficiency involve more than a dozen types of cancer including colon, breast and prostate, as well as the classic bone diseases: rickets, osteoporosis and osteomalacia. Irradiation with artificial UV sources can prevent the vitamin D deficiency. However, in view of different irradiation spectra of UV lamps, their ability to initiate vitamin D synthesis is different. The reliable method based on an in vitro model of vitamin D synthesis has been developed for direct measurement in situ of the vitamin D synthetic capacity of artificial UV sources during a phototherapeutic procedure

The aim is to study the effectiveness of continuous Nd:YAG laser inhibiting caries progression of human enamel. After surface polishing, enamel blocks irradiated by different power laser and control group were put into 5ml of lactate buffer solution (0.1mol/L), which was in water of 37°C for 72 hours. The microhardness and morphology of enamel surface were investigated by microhardness tester and scanning electron microscopy. Compared with control, the group with 0.3W laser treatment has the least caries lesion (p<0.001). At the same time, the mechanism of caries prevention and acid resistance by laser was discussed in detail. We drew a conclusion that the change of contents in enamel and crystal orientations and partial decomposition of the organic matrix might be the main reason for enhancing the acid resistance ability of enamel. And best caries inhibition could be achieved when the temperature rising of enamel surface was in the range of 300~400°C by laser parameters being controlled suitably.

Optical Coherence Tomography is a powerful, noninvasive biomedical technique that uses low coherence light sources to obtain in-depth scans of biological tissues. In this study, we report results obtained with three different sources: a 60 nm bandwidth superluminescent diode with a 1570 nm emission wavelength, a high power broadband fiber source (up to 100 nm bandwidth around 1330 nm wavelength), and a Ti:Sapphire ultrashort-pulsed laser (810 nm emission wavelength and 100 nm maximum bandwidth). Along with enhancement of some details and discontinuities in heterogeneous tissues, characterization of samples using these three wavelengths allows for a more complete description of tissue optical properties, such as attenuation, backscattering, or penetration depth. We will present results obtained in vitro on several samples of biological tissues.

Enhanced green fluorescent protein (EGFP)-expressing cells are customarily used in a variety of in vitro and in vivo studies and assays to ease visualization and localization. Nonetheless, the effects of EGFP expression on cellular responsivity to Photodynamic therapy (PDT), a combination therapy combining a photoactive drug and light, have yet to be characterized. To address this effect, rat astrocytoma cells (CNS-1), a lentivirus-transduced EGFP variant (CNS-1 GFP), human glioblastoma (U-87), and the transfected EGFP variant (U-87 GFP) are analyzed in terms of cell survival following PDT mediated by two different photoactive drugs. Cell survival is quantified via colony forming assays and Alamar blue assays, as a function of light dose, using the photosensitizers Photofrin (1ug ml^-1 for 24h) and ALA (200ug ml^-1 for 5h). Furthermore, effect of GFP expression on the responsivity to Cisplatin, a DNA-binding chemotherapeutic agent is determined for these cell lines. Our results show that EGFP expression does not affect the responsivity of Photofrin-PDT in comparison to parental cell lines (non GFP expressing cells), but does alter that of ALA-PDT. No change in responsivity is observed for Cisplatin treatment for either cell line. These results can be explained by oxidative stress induced by EGFP expression. This work will establish under which circumstances it is appropriate to use EGFP-expressing cell lines in the context of PDT preclinical research in vivo and in vitro.

A technique to produce fluorescent cell phantom standards based on calcium alginate microspheres with encapsulated fluorescein-labeled dextrans is presented. An electrostatic ionotropic gelation method is used to create the microspheres which are then exposed to an encapsulation method using poly-l-lysine to trap the dextrans inside. Both procedures were examined in detail to find the optimal parameters producing cell phantoms meeting our requirements. Size distributions favoring 10-20 microns microspheres were obtained by varying the high voltage and needle size parameters. Typical size distributions of the samples were centered at 150 μm diameter. Neither the molecular weight nor the charge of the dextrans had a significant effect on their retention in the microspheres, though anionic dextrans were chosen to help in future capillary electrophoresis work. Increasing the exposure time of the microspheres to the poly-l-lysine solution decreased the leakage rates of fluorescein-labeled dextrans.

This paper addresses the optimization of power at the circuit level in the main blocks of CMOS APS image sensors. A pixel bias current of zero during the readout period is shown to reduce the static power and enhance the settling time of the pixel. A balanced operational transconductance amplifier (OTA) has been demonstrated to be a better candidate as an amplifier when employed in a correlated double sampling (CDS) circuit or as a comparator in an analog-to-digital (A/D) converter, as compared to a Miller two-stage amplifier. Using common-mode feedback (CMFB) in an OTA can further reduce the quiescent power of the amplifier. The low power capability of a CMFB OTA is discussed in this paper by performing a comparison with a conventional OTA using a 0.18 μm technology.

Kernel-based pattern recognition paradigms such as support vector machines (SVM) require computationally intensive feature extraction methods for high-performance real-time object detection in video. The CMOS sensory parallel processor architecture presented here computes delta-sigma (ΔΣ)-modulated Haar wavelet transform on the focal plane in real time. The active pixel array is integrated with a bank of column-parallel first-order incremental oversampling analog-to-digital converters (ADCs). Each ADC performs distributed spatial focal-plane sampling and concurrent weighted average quantization. The architecture is benchmarked in SVM face detection on the MIT CBCL data set. At 90% detection rate, first-level Haar wavelet feature extraction yields a 7.9% reduction in the number of false positives when compared to classification with no feature extraction. The architecture yields 1.4 GMACS simulated computational throughput at SVGA imager resolution at 8-bit output depth.

Detailed knowledge of protein structure is vital to the understanding of numerous biological processes and to expedite advancements in pharmaceutical research. The atomic structure of protein can be determined by measuring the intensity of its X-ray diffraction pattern. The functional wavelength of X-ray for this purpose lies in the range of 0.01-1 nm. The diffraction pattern produced by X-ray of such wavelength can be read by a large area (~ 20 cm x 20 cm) detector. The detector should have good spatial resolution (FWHM ~ 2 pixels) to detect every Bragg peak in the pattern. In addition, a wide dynamic range (~10⁶) is desirable to accurately measure the intensity of Bragg peaks. Amorphous silicon (a-Si:H) based detector is a potential candidate to meet these requirements; in particular, it is attractive by virtue of its inexpensive manufacturing process and large area compatibility compared to the existing CCD and image plate based detection techniques. In this work, we investigate by modeling the feasibility of an a-Si:H based detector for the study of protein structure. The proposed detector employs amorphous selenium (a-Se) photoconductor layer to directly convert the incident X-ray to a charge image, which is then electronically read by an array of a-Si:H thin film transistors. The modeling results show that the detector reasonably satisfies the requirements for determination of protein.

In this paper, an innovative current-programmed, current-output active TFT image sensor suitable for real time x-ray imaging (fluoroscopy) using hydrogenated amorphous silicon (a-Si:H) thin film transistor (TFT) technology coupled with a transimpedance feedback column amplifier for pixel signal readout is presented. Simulation results show that this new TFT circuit can successfully compensate for variations in a-Si:H TFT characteristics under prolonged gate voltage stress. The readout is fast enough to fulfill the timing requirements of digital fluoroscopy. Dynamic effects such as charge injection, charge feed-through and drain-source voltage variation as well as additive noise of the pixel TFTs induce error on the output current of the pixel. To explore the dependence of this error on pixel parameters, concise analytical expressions are derived which can be used to reduce the amount of the output current error by proper pixel design.

The human vision system (HVS) is remarkably robust against eye distortions. Through a combination of eye movements and visual feedback, the HVS can often appropriately interpret scene information acquired from flawed optics. Inspired by biological systems, we have built an electronically and mechanically reconfigurable "saccadic" camera system. The saccadic camera is designed to efficiently examine scenes through foveated imaging, where scrutiny is reserved for salient regions of interest. The system's "eye" is an electronic image sensor used in multiple modes of resolution. We use a subwindow set at high resolution as the system's fovea, and capture the remaining visual field at a lower resolution. The ability to program the subwindow's size and position provides an analog to biological eye movements. Similarly, we can program the system's mechanical components to provide the "neck's" locomotion for modified perspectives. In this work, we use the saccadic camera to develop a "work-around" routine in response to possible degradations in the camera's lens. This is particularly useful in situations where the camera's optics are exposed to harsh conditions, and cannot be easily repaired or replaced. By exploiting our knowledge of the image sensor's electronic coordinates relative to the camera's mechanical movement, the system is able to develop an empirical distortion model of the image formation process. This allows the saccadic camera to dynamically adapt to changes in its image quality.

For objects on a plane, a "scale factor" relates the physical dimensions of the objects to the corresponding dimensions in a camera image. This scale factor may be the only calibration parameter of importance in many test applications. The scale factor depends on the angular size of a pixel of the camera, and also on the range to the object plane. A measurement procedure is presented for the determination of scale factor to high precision, based on the translation of a large-area target by a precision translator. A correlation analysis of the images of a translated target against a reference image is used to extract image shifts and the scale factor. The precision of the measurement is limited by the translator accuracy, camera noise and various other secondary factors. This measurement depends on the target being translated in a plane perpendicular to the optic axis of the camera, so that the scale factor is constant during the translation. The method can be extended to inward-looking 3D camera networks and can, under suitable constraints, yield both scale factor and transcription angle.

With the aid of a differential polarization (DP) apparatus, developed in our laboratory and attached to our laser scanning confocal microscope, we can measure the magnitude and spatial distribution of 8 different DP quantities: linear and circular dichroism (LD&CD), linear and circular anisotropy of the emission (R and CPL, confocal), fluorescence detected dichroisms (FDLD&FDCD, confocal), linear birefringence (LB), and the degree of polarization of fluorescence emission (P, confocal). The attachment uses high frequency modulation and subsequent demodulation, via lock-in amplifier, of the detected intensity values, and records and displays pixel-by-pixel the measured DP quantity. These microscopic DP data carry important physical information on the molecular architecture of anisotropically organized samples. Microscopic DP measurements are thought to be of particular importance in biology. In most biological samples anisotropy is difficult to determine with conventional, macroscopic DP measurements and microscopic variations are of special significance. In this paper, we describe the method of LB imaging. Using magnetically oriented isolated chloroplasts trapped in polyacrylamide gel, we demonstrate that LB can be determined with high sensitivity and good spatial resolution. Granal thylakoid membranes in edge-aligned orientation exhibited strong LB, with large variations in its sign and magnitude. In face-aligned position LB was considerably weaker, and tended to vanish when averaged for the whole image. The strong local variations are attributed to the inherent heterogeneity of the membranes, i.e. to their internal differentiation into multilamellar, stacked membranes (grana), and single thylakoids (stroma membranes). Further details and applications of our DP-LSM will be published elsewhere.

Optical coherent tomography (OCT) is a newly developed optical imaging technology that permits high-resolution cross-sectional imaging of an object. Most of the OCT imaging systems is developed for the biomedical applications, such as diagnostics of ophthalmology, dermatology, dentistry and cardiology. The technique behind these applications is the point scanning of laser beam penetrating into an object to obtain the internal features of the object. In this paper, we study a full-field OCT imaging system for acquiring information from a multi-layer information chip. This new system can be used in document security, identification and industrial inspection. Differing from the biology related samples, the information chip consists of a number of thin layers with information coded on their surfaces. The surfaces of the layers are flat and specular with moderate reflectance. The information on one layer is retrieved through demodulating interference image of that layer. To obtain the tomography image of all the layers, the images in each layer are acquired and separated. The axial resolution of the system, usually defined by coherent length of light source, determines how close the separation of the two vicinal layers can be resolved. In this paper we explore a new approach to enhance the axial resolution of the OCT system. The method is based on a three-step phase shift algorithm to solve the tomography images from fused interference patterns. Theoretic study and simulation indicate that the method improves the system resolution and quality of retrieved tomography images for the multilayer information chip. Experiment results are also given in support of the proposed method.

The use of digital fluorescence confocal microscopy in biological sciences has grown in recent decades due to the versatility of fluorescence imaging. The ability to selectively label specific morphological features, genetic mutations and/or chemical micro-environmental changes with discreet fluorescent labels allows a better understanding of the complex systems that regulate cellular processes. Specimens can range in size from single cells to tissue sections and tissue arrays, which can occupy the entire surface of a microscope slide (25mm x 70mm). Using a confocal scanning laser MACROscope, a wide-area confocal imaging system (Biomedical Photometrics Inc.), it is possible to image these large specimens at high resolution, without the need to tile many small microscope fields. A hyperspectral imaging (HSI) mode has been added to the MACROscope system to assess the use of HSI in the removal/separation of tissue autofluorescence from digital images of fluorescently-labeled paraffin-embedded, formalin-fixed tissue sections. In pathology and immunohistochemistry applications this autofluorescence can hinder, or even prevent, detection of the applied fluorescent label(s). In the present study, fluorescence emission from the specimen was sampled at ~7 nm bandwidths across 32 channels, amounting to viewing ~220 nm of the visible spectrum as a hyperspectral data cube. The data cube was then processed to remove the contributions from autofluorescence, leaving only the signal from the fluorophore(s) of interest. Comparisons are drawn from HSI obtained with a commercial hyperspectral confocal microscope (Zeiss LSM 510 META) employing image tiling. The initial results demonstrate the ability to spectrally unmix the tissue autofluorescence in large tissue sections.

Our newly developed multimodal microscope enables simultaneous collection of second harmonic generation (SHG), third harmonic generation (THG) and multiphoton excitation fluorescence (MPF) signals. The signals can be generated within different or the same intercellular structures. In comparing two signals, traditional methods of image crosscorrelation analysis using Pearson's coefficient provide a general parameter as to whether the images are similar, however it does not give detailed information about correlation of different structures inside the images. We present here a new technique that employs a pixel by pixel analysis over an entire area or volume that is used to correlate the structures appearing in the images. The result of the analysis reveals structures within the sample that are generated by both nonlinear signals as well as highlighting the structures that are generated by only one of the nonlinear signals. The algorithm provides a means to colocalize different structures revealed by the different nonlinear contrast mechanisms. Structural correlation maps are useful in identifying the origin of structures in one nonlinear contrast mechanism when the origin of structures in another is known. Image analysis has also been exploited for sequences of images taken in time. The intensity fluctuations in time for each pixel reveal regions of intense physiological activity in biological samples. Correlation of time dependent fluctuations from different pixels in the image time series allows construction of the structural map that undergoes similar time behavior or appears out of phase. These structural correlation analysis techniques are demonstrated based on polystyrene beads and cardiomyocytes.

The ability to watch atoms move in real time - to directly observe transition states - has been referred to as "making the molecular movie". Femtosecond electron diffraction is ideally suited for this purpose since it records the atomic structure of the sample with sub-Angstrom spatial resolution and femtosecond temporal resolution. Many-body simulations of ultrashort electron pulse propagation dynamics allowed the development of sources for femtosecond electron pulses with sufficient number density to perform near single shot structure determinations, a requirement for studies of irreversible processes. We have obtained atomic level views of melting of thin films of aluminum and gold under strongly driven conditions. The results are consistent with a thermally driven phase transition and the observed time scales reflect the different electron-phonon coupling constants for these metals. Recent technical advances in electron gun design have further improved the temporal resolution of femtosecond electron diffraction. New electron pulse characterization techniques use direct laser-electron interaction and electron-electron interaction to determine the temporal overlap of the pump and probe pulses as well as the time resolution of the system. These advances have made femtosecond electron diffraction capable of observing transition states in molecular systems. The camera for "making the molecular movie" is now in hand.

We previously developed a Mueller matrix formalism to improve confocal imaging in microscopes and ophthalmoscopes. Here we describe a procedure simplified by firstly introducing a generator of polarization states in the illumination pathway of a confocal scanning laser microscope and secondly computing just four elements of the Mueller matrix of any sample and instrument combination. Using a subset of Mueller matrix elements, the best images are reconstructed. The method was tested for samples with differing properties (specular, diffuse and partially depolarizing). Images were also studied of features at the rear of the eye. The best images obtained with this technique were compared to the original images and those obtained from frame averaging. Images corresponding to non-polarized incident light were also computed. For all cases, the best reconstructed images were of better quality than both the original and frame-averaged images. The best reconstructed images also showed an improvement compared with the images corresponding to non polarized light. This methodology will have broad application in biomedical imaging.

Air borne sensed data is in the form of raster data. Aliasing is always present in a sampled image causing artifact error. To reduce possible aliasing effects, it is a good idea to blur an image slightly before applying a resampling method on it. This paper presents a technique for anti-aliasing air borne sensed images. The technique uses Gaussian low pass filter (GLPF) for generation of slight blur and reduction of high frequency components. Then resampling of raster data is performed with the help of bilinear interpolation. Algorithm is developed in MATLAB using some inbuilt functions. The method is applied on different types of images, and their frequency spectrum and histogram analysis is carried out.

The goal of this work is to develop a simple and systematic method to highlight the properties of filters for their application in temporal phase shifting interferometry. In this study, the effects of elementary filters (mean, gaussian and median masks) are analyzed. In order to compare those filters, correlation fringes were numerically synthesized and a Gaussian noise has been added. The advantages and the failures of each studied filtering mask have been enhanced thanks to the comparison of different profiles and fidelity functions. Finally, this study is applied to the filtering of a shearogram recorded in our laboratory.

The dynamic range in many real-world environments surpasses the capabilities of traditional display technologies by several orders of magnitude. Recently, a novel display capable of displaying images with a dynamic range much closer to real world situations has been demonstrated. This was achieved through a spatially modulated backlight behind an LCD panel. Combined with the modulating power of the LCD panel itself, this enabled the display of much higher contrast compared to an LCD panel with a spatially uniform backlight. In this paper, we describe a further development of the technology, namely a high dynamic range projection system. This makes such display systems more widely applicable as any surface can be used for the display of high dynamic range images. Our new system is designed as an external attachment to a regular DLP^TM-based projector, which allows the use of unmodified projectors. It works by adapting the projected image via a set of lenses to form a small image. This small image is then modulated via an LCD panel and the result is projected via another lens system onto a larger screen, as in traditional projection scenarios. The double modulation, by the projector and the LCD panel together, creates a high dynamic range image and an ANSI contrast of over 700:1. Finally, we discuss the advantages and disadvantages of our design relative to other high and low dynamic range display technologies and its potential applications.

With the outbreak of Bovine Spongiform Encephalopathy (BSE) (commonly known as mad cow disease) in 1987 in the United Kingdom and a recent case discovered in Alberta, more and more emphasis is placed on food and farm feed quality and safety issues internationally. The disease is believed to be spread through farm feed contamination by animal byproducts in the form of meat-and-bone-meal (MBM). The paper reviewed the available techniques necessary to the enforcement of legislation concerning the feed safety issues. The standard microscopy method, although highly sensitive, is laborious and costly. A method to routinely screen farm feed contamination certainly helps to reduce the complexity of safety inspection. A hyperspectral imaging system working in the near-infrared wavelength region of 1100-1600 nm was used to study the possibility of detection of ground broiler feed contamination by ground pork. Hyperspectral images of raw broiler feed, ground broiler feed, ground pork, and contaminated feed samples were acquired. Raw broiler feed samples were found to possess comparatively large spectral variations due to light scattering effect. Ground feed adulterated with 1%, 3%, 5%, and 10% of ground pork was tested to identify feed contamination. Discriminant analysis using Mahalanobis distance showed that the model trained using pure ground feed samples and pure ground pork samples resulted in 100% false negative errors for all test replicates of contaminated samples. A discriminant model trained with pure ground feed samples and 10% contamination level samples resulted in 12.5% false positive error and 0% false negative error.

Semiconductor devices that are not generally thought of as light sources do emit radiation in the visible and the near infrared as they operate. Observation of this electroluminescence furnishes insight into the operation of the devices and of the circuitry that they constitute but it requires an extremely sensitive light detector and picosecond time resolution. This can be achieved using a Mepsicron photodetector system that enables single photon counting time-correlated imaging with a spatial resolution of about 1 µm and a time resolution approaching 10 ps. Information extracted from the time-resolved imagery can be compared with circuit layout and topology and individual device structures and with electrical measurements that are performed concurrently. These time-correlated measurements allow signal waveforms to be determined optically, much as the waveforms measured electronically with an oscilloscope and microprobing, but with the advantage that the acquisition is entirely non-invasive. Images can be dissected in both space and time to provide information for individual components of a circuit or regions of a device. This imaging equipment has been used in our laboratory for measurements on Si, GaAsP and GaN technologies and analyses will be presented.