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Trans–cis–trans isomerization laser spectroscopy is combined with DFT theoretical calculations, to measure and model the geometric inter-conversion behaviour of the much-used Bismarck Brown Y azo dye.

Radiochromic material used in recent commercial films has been suggested as a candidate for in vivo dosimetry because of its dose sensitivity, real-time response, and atomic composition. It was observed that its sensitive material, lithium pentacosa-10,12-diynoate (LiPCDA), can have two distinct forms, with main absorbance peaks at ∼635 and ∼674 nm. The spectrum of the latter is similar to that of pentacosa-10,12-diynoic acid (PCDA) used in the commercial predecessor, obtained through desiccation of the commercial film. Water was suggested to be a part of the crystal structure and thus its presence or absence would affect dosimetric parameters. The objective of this study is to: (a) investigate how desiccated commercial films compared to the native form in terms of macroscopic crystal structure, dose-response, signal linearity, and post-exposure kinetics; (b) demonstrate proof-of-concept that the two versions can be combined into one optical dosimeter and measured simultaneously.Commercial radiochromic film, EBT-3, was desiccated for 10 days at 45°C. Using a 6 MV LINAC beam and standard setup of 100 Source to Axis Distance (SAD), 10 cm × 10 cm field size, and 1.5 cm depth, commercial and desiccated films were irradiated to 50, 100, 200, 500, 1000, 2000, 3000 cGy and the latter to 4000, 5000, and 7000 cGy. A custom phantom equipped with optical fibers for real-time read-out was used for all measurements. Absorbance spectra were collected at ∼1 Hz before, during, and after irradiation. Data were collected for ∼1 h after the end of irradiation for 200 cGy experiments. The radiation-induced change in optical density (∆OD) was calculated with a 10 nm band around the primary absorbance peak. The post-exposure percent optical density change was calculated and compared to ∆OD at the end of irradiation. Both commercial and desiccated films were also irradiated and measured simultaneously as proof-of-concept for using two materials within one optical path. For electron microscopy imaging, active materials from commercial and desiccated films were imaged on a scanning electron microscope at an accelerating voltage of 10 kV.Scanning electron microscope images showed that desiccated film was similar in topographical structure to the commercial EBT-3 form. It maintained a non-linear ∆OD with dose but resulted in ∼1/3 signal compared to the commercial film. Evaluation of post-exposure response showed significantly lower percent increase in ∆OD for desiccated film initially, with no statistically significant difference at 1 h after the end of irradiation. Combining both films and simultaneously measuring their absorbance illustrated that the two absorbance peaks were identifiable and resolvable to allow for an independent determination of dose from each.Water is implicated in the crystal structure of the EBT-3 radiochromic film, with its removal through desiccation affecting both dosimetric and spectroscopic characteristics of the material. The two forms of radiochromic material (with and without water) are spectrally resolvable allowing for independent dose determination from each, opening up possibilities for dose measurements at different locations along a single fiber.

We study here the role of polyelectrolyte chain length, that is number of repeat units (mers), in the competitive adsorption of a simple model polyanion, poly(acrylic acid), onto 85 nm spherical silica particles capped with a model polycation, poly(allylamine hydrochloride). Performing fluorescence spectroscopy experiments, we measured chain-length dependence of dilute aqueous polyelectrolyte adsorption, at full surface coverage, onto an oppositely charged polyelectrolyte overtop spherical silica nanoparticles (10-3 g L-1). Preferential adsorption was determined by comparing the characteristic fluorescence intensities of the two fluorophore-labeled and narrowly disperse polyacrylic acid samples (NMA-PAA450k and Dan-PAA2k) of 450k- and 2k-molecular weight (6250- and 28-mers), respectively. To compare and validate experimental results, a lattice model was developed for computing the probabilities of the different arrangements of two polymer chain lengths of polyacrylic acid on the surface of the silica nanosphere. We then determined which numbers of long and short adsorbed chains corresponded to the most configurations in our model. Both spectroscopic experiment results and the combinatorial model demonstrated that there is an entropic preference for complete adsorption of the longer 450k polyacrylic acid chain vs. 2k. This study provides insights on entropy driven chain-length dependence of polyelectrolyte adsorption onto spherical nanoparticle surfaces for directing and optimizing their layer-by-layer self-assembly in organic films.

Photoacoustic sensing can be a powerful technique to obtain real-time feedback of laser energy dose in treatments of biological tissue. However, when laser therapy uses pulses with microsecond duration, they are not optimal for photoacoustic pressure wave generation. This study examines a programmable fiber laser technique using pulse modulation in order to optimize the photoacoustic feedback signal to noise ratio (SNR) in a context where longer laser pulses are employed, such as in selective retinal therapy. We have demonstrated with a homogeneous tissue phantom that this method can yield a greater than seven-fold improvement in SNR over non-modulated square pulses of the same duration and pulse energy. This technique was further investigated for assessment of treatment outcomes in leporine retinal explants by photoacoustic mapping around the cavitation-induced frequency band.

Pilot results showing the potential of sub-microsecond laser pulse shaping to optimize thermomechanical confinement in laser-tissue interactions involving microcavitation are presented. Model samples based on aqueous suspensions of retinal melanosomes and eumelanin particles were irradiated at 532 nm with nanosecond laser pulses and picosecond laser pulse trains having differing shapes and durations. The cavitation threshold radiant exposure and the bubble lifetime above the threshold were measured using a pump-probe setup and sub-nanosecond time-resolved imaging. Both quantities were found to strongly depend on the pulse format. These results suggest that sub-microsecond laser pulse shaping could be exploited to optimize precision and control in numerous applications of laser-directed microcavitation, including selective retinal laser treatments.

Lasers are employed for numerous medical interventions by exploiting ablative, disruptive or thermal effects. In ocular procedures, lasers have been used for decades to treat diseases such as diabetic retinopathy, macular edema and aged related macular degeneration via photocoagulation of retinal tissues. Although laser photocoagulation is well established in today’s practice, efforts to improve clinical outcomes by reducing the collateral damage from thermal diffusion is leading to novel treatments using shorter (μs) laser pulses (e.g. selective retinal therapy) which result in physical rather than thermal damage. However, for these new techniques to be widely utilized, a method is required to ensure safe but sufficient dosage has been applied, since no visible effects can be seen by ophthalmoscopy directly post treatment. Photoacoustic feedback presents an attractive solution, as the signal is dependent directly on absorbed dosage. Here, we present a method that takes advantage of temporal pulse formatting technology to minimize variation in absorbed dose in ophthalmic laser treatment and provide intelligent dosimetry feedback based on photoacoustic (PA) response. This method tailors the pulse to match the frequency response of the sample and/or detection chain. Depending on the system, this may include the absorbing particle size, the laser beam diameter, the laser pulse duration, tissue acoustic properties and the acoustic detector frequency response. A significant improvement (

Optical biopsy of tissue using fiber optic probes has proven to be a powerful tool for non-invasive and minimally invasive diagnostics. However, there are still many challenges to improving diagnostic value and commercial translation of these techniques. Many fiber-based methods are limited by background noise, which impairs sensitivity and specificity. Aspects of quality control, such as adequacy of the target of interest sampled and validation of optical measurements with histopathology can be problematic. Complexity, cost, and disposability or sterilizability are roadblocks to widespread clinical use. Here, we present new approaches to using fibers for optical biopsy aimed at solving these problems. Specifically, the new concepts are designed with the goals of being simple and disposable, to improve control of light delivery and collection from the sample, and to inherently enable better quality control of the biopsy process. A concept-of-operation aimed at nearly zero impact to the work flow of the biopsy and standard pathology procedures will be outlined. Several concepts for fiber implementations will be presented. A trade-off analysis of the concepts used to select a first implementation for testing will be presented. Preliminary experimental validation in phantoms and tissue samples will be presented for the selected configuration.

A fiber-optic based flow cytometry platform was designed to build a portable and robust instrument for space applications. At the core of the Microflow1 is a unique fiber-optic flow cell fitted to a fluidic system and fiber coupled to the source and detection channels. A Microflow1 engineering unit was first tested and benchmarked against a commercial flow cytometer as a reference in a standard laboratory environment. Testing in parabolic flight campaigns was performed to establish Microflow1's performance in weightlessness, before operating the new platform on the International Space Station. Microflow1 had comparable performances to commercial systems, and operated remarkably and robustly in weightlessness (microgravity). Microflow1 supported immunophenotyping as well as microbead-based multiplexed cytokine assays in the space environment and independently of gravity levels. Results presented here provide evidence that this fiber-optic cytometer technology is inherently compatible with the space environment with negligible compromise to analytical performance.

Selective retinal therapy is a promising alternative to laser photocoagulation in the treatment of several retinal diseases. It uses short microsecond laser pulses causing cavitation(s) and photo-mechanical damage to the highly absorptive retinal pigment epithelium (RPE) cells. Here, we investigate the effect of laser temporal pulse shaping (ps-burst vs continuous) on the cavitation dynamics in RPE tissue.

The capacity to engineer the extracellular matrix is critical to better understand cell function and to design optimal cellular environments to support tissue engineering, transplantation and repair. Stacks of adsorbed polymers can be engineered as soft wet three dimensional matrices, with properties tailored to support cell survival and growth. Here, we have developed a combinatorial method to generate coatings that self assemble from solutions of polyelectrolytes in water, layer by layer, to produce a polyelectrolyte multilayer (PEM) coating that has enabled high-throughput screening for cellular biocompatibility. Two dimensional combinatorial PEMs were used to rapidly identify assembly conditions that promote optimal cell survival and viability. Conditions were first piloted using a cell line, human embryonic kidney 293 cells (HEK 293), and subsequently tested using primary cultures of embryonic rat spinal commissural neurons. Cell viability was correlated with surface energy (wettability), modulus (matrix stiffness), and surface charge of the coatings.Our findings indicate that the modulus is a crucial determinant of the capacity of a surface to inhibit or support cell survival.

The surface molecular structure and adsorbed mass of a series of model amphiphilic peptides have been studied in situ with surface-specific sum frequency generation (SFG) vibrational spectroscopy and quartz crystal microbalance (QCM) at the hydrophobic polystyrene and hydrophilic silica solid−water interface. The peptides are designed to form α-helical (XY14) and β-strand (XY7) secondary structures at an apolar interface and contain hydrophobic (X) and charged (Y) amino acids with sequence Ac-XYYXXYXXYYXXYX-NH2 or Ac-XYXYXYX-NH2, respectively. The X and Y combinations are leucine (L) and lysine (K), alanine (A) and lysine (K), alanine (A) and arginine (R), and phenylalanine (F) and arginine (R). One additional peptide with sequence Ac-LKKLLKL-NH2 (LK7 α) was synthesized. These peptides allow for the study of how chain length (LK14 vs LK7 α and LK7 β), amino acid side chain character (LK vs AK vs AR vs FR), and sequence (LK7 α-helix vs LK7 β-strand sequences) affect adsorption. The SFG spectra of adsorbed ...

The evolution of biological surface science can be credited to the development of traditional surface-chemistry tools and techniques to investigate molecular and atomic-scale bonding, structure, conformation, physical properties (e.g., chemical, electronic, mechanical), and dynamics of adsorbates at various interfaces:1 Both classical measurements of surface behavior and features (i.e., adsorption isotherms, surface areas, roughness, thickness, and topography) and modern spectroscopic-based techniques that provide information on elemental composition, oxidation state, depth profiling, and distribution of chemical species have shown applicability to the study of biomolecular interactions.1 However, experiments that probe with electrons, atoms or ions require ultrahigh vacuum (UHV) or reduced pressures at the interface, and are thus intrinsically limited with regards to interfacial explorations in an aqueous environment, i.e., the study of at biomolecules the solid/water interface.1

Azobenzene chromophores can be switched between two geometric isomers using visible light. This photoisomerization is rapid, reversible, and of high quantum yield, and the wavelengths effecting the transformation can be tuned synthetically with substituent groups to the chromphores. Upon isomerization, there can be significant changes to the optical, geometric, mechanical, and chemical properties of azobenzene molecules, and these photo-switchable properties can often be transferred to large host systems into which azobenzene is incorporated. This review describes polymers and surfaces that have been prepared recently that incorporate azobenzene groups, and some of the interesting physical and chemical properties that can be switched reversibly as a result.

Mechanical properties of polyelectrolyte multilayer thin films were studied by nanoinden- tation experiments using an atomic force microscope. We obtained force-distance measurements for a model polycation/polyanion multilayer system, poly(allylamine hydrochloride) (PAH), and an azobenzene- containing polyelectrolyte (P-Azo) prepared at varying charge densities. The relative Young's modulus of the films was determined as a function of the ionization fraction of the multilayer films, prepared to have identical thickness. Multilayer films assembled with polyelectrolytes of high charge density exhibited an elastic modulus that was significantly larger (nearly 2 orders of magnitude) than those prepared with low charge density. An estimate of the relative loop length between "ionic cross-links" in the multilayer films is then ascertained by analogy to previously studied covalently cross-linked polymer networks. The modulus values in our films ranged from 10-2 to 10-4 kPa, and this implies loop lengths of 1-50 segments. Atomic force microscopy force-distance measurements were also used to compare the relative adhesion values between polycation and polyanion layers in films constructed with varying charge densities. This was done by coating an AFM tip with multilayers and indenting into a surface containing the same multilayer film but capped with the oppositely charged surface polyelectrolyte layer. Adhesion values were typically between 0.5 and 6.7 nN and were found to depend on the ionic cross-link density of the PAH/P-Azo film, in which the highly ionically cross-linked samples exhibited the largest adhesion.

The kinetics of the electrostatic layer-by-layer adsorption of a weakly charged polycation, poly(allylamine hydrochloride), PAH, and a polyanion containing an azobenzene chromophore, P-Azo, was studied using UV−vis spectroscopy and ellipsometry. The thickness of the multilayer films was first measured over the adsorption pH range of 3 to 11, and the growth of multilayers was examined as function of time and concentration. Films assembled in bath pH near that of their pKa value produced both the thickest films and displayed remarkably rapid adsorption isotherms. In some PAH/P-Azo films, a significantly large thickness was achieved in less than 5 s, which is more than 2 orders of magnitude faster than what is usually observed. We show that this anomalously rapid adsorption is a consequence of the weak acid−base nature of the layers. In addition, the role of counterion displacement in multilayer formation from weakly charged polyelectrolytes was explored by examining the rates of P-Azo adsorption in PAH/P-Az...

The translation of CARS imaging towards real time, high resolution, chemically selective endoscopic tissue imaging applications is limited by a lack of sensitivity in CARS scanning probes sufficiently small for incorporation into endoscopes. We have developed here a custom double clad fiber (DCF)-based CARS probe which is designed to suppress the contaminant Four-Wave-Mixing (FWM) background generated within the fiber and integrated it into a fiber based scanning probe head of a few millimeters in diameter. The DCF includes a large mode area (LMA) core as a first means of reducing FWM generation by ~3 dB compared to commercially available, step-index single mode fibers. A micro-fabricated miniature optical filter (MOF) was grown on the distal end of the DCF to block the remaining FWM background from reaching the sample. The resulting probe was used to demonstrate high contrast images of polystyrene beads in the forward-CARS configuration with > 10 dB suppression of the FWM background. In epi-CARS geometry, images exhibited lower contrast due to the leakage of MOF-reflected FWM from the fiber core. Improvements concepts for the fiber probe are proposed for high contrast epi-CARS imaging to enable endoscopic implementation in clinical tissue assessment contexts, particularly in the early detection of endoluminal cancers and in tumor margin assessment.

A multi-center study has been set up to accurately characterize the optical properties of diffusive liquid phantoms based on Intralipid and India ink at near-infrared (NIR) wavelengths. Nine research laboratories from six countries adopting different measurement techniques, instrumental set-ups, and data analysis methods determined at their best the optical properties and relative uncertainties of diffusive dilutions prepared with common samples of the two compounds. By exploiting a suitable statistical model, comprehensive reference values at three NIR wavelengths for the intrinsic absorption coefficient of India ink and the intrinsic reduced scattering coefficient of Intralipid-20% were determined with an uncertainty of about 2% or better, depending on the wavelength considered, and 1%, respectively. Even if in this study we focused on particular batches of India ink and Intralipid, the reference values determined here represent a solid and useful starting point for preparing diffusive liquid phantoms with accurately defined optical properties. Furthermore, due to the ready availability, low cost, long-term stability and batch-to-batch reproducibility of these compounds, they provide a unique fundamental tool for the calibration and performance assessment of diffuse optical spectroscopy instrumentation intended to be used in laboratory or clinical environment. Finally, the collaborative work presented here demonstrates that the accuracy level attained in this work for optical properties of diffusive phantoms is reliable. © 2014 Optical Society of America.

Proof of concept results are presented towards an in situ bimodal proximity sensor for neurovascular bundle detection during dental implant surgery using combined near infrared absorption (NIR) and optical coherence tomography (OCT) techniques. These modalities are shown to have different sensitivity to the proximity of optical contrast from neurovascular bundles. NIR AC and DC signals from the pulsing of an artery enable qualitative ranging of the bundle in the millimeter range, with best sensitivity around 0.5-3mm distance in a custom phantom setup. OCT provides structural mapping of the neurovascular bundle at sub-millimeter distances in an ex vivo human jaw bone. Combining the two techniques suggests a novel ranging system for the surgeon that could be implemented in a “smart drill.” The proximity to the neurovascular bundle can be tracked in real time in the range of a few millimeters with NIR signals, after which higher resolution imaging OCT to provide finer ranging in the sub-millimeter distances.

We report an innovative label-free biosensor based on statistical analysis of several whispering gallery modes spectral shifts in polystyrene fluorescent microspheres using a custom microflow cytometer. Whispering gallery modes analysis enables detection of nanometer-sized analytes showing promising possibilities for virus, bacteria and molecular detection. To demonstrate this, fluorophore-doped microspheres of the appropriate size parameter are mixed in an aqueous solution. Then, a syringe pump pushes the solution through a fiber optic flow cell where a laser beam illuminates the analysis area to excite the microspheres and their fluorescence is collected. This device provides a low-cost and user friendly solution that could enhance spectrum acquisition rates up to 5 spectra per second thanks to the considerable amount of microspheres flowing through the excitation area per unit time. Finally, the fluorescence spectra are statistically investigated using an instantaneous measurement of apparent refractive index algorithm to determine a reliable value for the refractive index of the environment since the exact radius of the microsphere scanned is unknown. This refractive index becomes an effective value for the local perturbation caused by inhomogeneities on the microsphere surface and hence, determines whether or not inhomogeneities, such as bacteria, are adsorbed by comparing to a control sample. Combining a flow cell with our detection algorithm, we reduce the period of a 50 microspheres experiment from 161 minutes to 14 minutes when the flow rate is 2000 µl/h and the microsphere concentration is 5 µsphere/µl.

Based on an innovative in-vivo optical dosimeter platform developed by scientists at University Health Network, we miniaturized the optical dosimeter in a tiny probe that fits the tip of an optical fiber. The approach consists in a measure of the absorbance change of a sensitive radiochromic material. The increase in absorbance is measured at a single wavelength and the linearly depends on the ionizing radiation dose. For compactness and design reasons, the proposed probe works in a reflective mode. A significant drawback when working with a reflective configuration is that reflections coming from splitter interfaces add to the signal and cause an apparent deviation from linearity. We studied the back reflections coming from a standard splitter and two custom made bifurcated optical fibers assemblies; 1) 7 fibers and 19 fibers. The 7 fibers connected to a 500 μm plastic optical fiber had the lowest reflection of 0.016% which was 3 times less than the 19 fibers and 100 times less than the standard splitter. An appropriate choice of the splitter was then imperative otherwise an under evaluation of the relative absorbance of −30% will happen.

Optical tissue phantoms can serve many needs encountered in the translational path between fundamental research and clinical acceptance. Each of these needs call for a different set of requirements on the phantom design. Earlier stage research will require the phantom to reproduce adequately the measurement challenges of the intended application. Phantoms used during the final verification and validation phase of a medical device seeking FDA clearance will focus more on stability, repeatability and traceability. Developing and producing phantoms meeting these quality requirements is a challenging task. Unlike MRI or CT, optical technologies will not reach clinical practice as versatile multipurpose imaging platforms but as a collection of application specific instruments. This variety in the instrumentation translates into very diverse requirements for phantoms and is somehow a barrier to the standardization of diffuse optical spectroscopy. One common point of all diffuse optical spectroscopy instrumentation is the need for calibration which can be served by simple homogeneous reference material. A general consensus on the metrology of the optical properties of such reference material is required before it can become generally accepted by the community.

With the increasing use of optical coherence tomography (OCT) in biomedical applications, robust yet simple methods for calibrating and benchmarking a system are needed. We present here a procedure based on a calibration object complemented with an algorithm that analyzes three-dimensional OCT datasets to retrieve key characteristics of an OCT system. The calibration object combines state-of-the-art tissue phantom material with a diamond-turned aluminum multisegment mirror. This method is capable of determining rapidly volumetric field-of-view, axial resolution, and image curvature. Moreover, as the phantom material mimics biological tissue, the system’s signal and noise levels can be evaluated in conditions close to biological experiments. We believe this method could improve OCT quantitative data analysis and help OCT data comparison for longitudinal or multicenter studies.

There is a pressing need for a low cost, passive optical fiber dosimeter probe for use in real-time monitoring of radiation dose delivered to clinical radiation therapy patients. An optical fiber probe using radiochromic material has been designed and fabricated based on the deposition of a radiochromic thin film on a dielectric mirror. Measurements of the net optical density vs. time before, during, and after irradiation at a rate of 500 cGy/minute to a total dose of 5 Gy were performed. Net optical densities increased from 0.2 to 2.0 for radiochromic thin film thicknesses of 2 to 20 µm, respectively. An improved optical fiber probe fabrication method is presented.

Optical tissue phantoms are very important tools for the development of biomedical imaging applications. They play an important role from proof of concept to clinical trials. Optical phantoms are often used as reference materials against which instruments results can be compared. It is therefore important that the optical properties of reference phantoms be measured in a manner that is traceable to the international system of units. SI traceability insures long term consistency of results and will therefore improve the effectiveness of diffuse optics research effort more effective by reducing unwanted variability in the data produced and shared by the community. The ultimate benefit of SI traceability is the reduction of variability in the data produced by novel diagnostic devices, which will in turn increase the statistical power of clinical trials aiming at validating their clinical usefulness. SI traceability, which implies uncertainty analysis, is also relevant to traceability aspects mandated by FDA's Good Manufacturing Practices.

Optical tissue phantoms are very important tools for the development of biomedical imaging applications. Optical phantoms are often used as ground truth against which instruments results can be compared. It is therefore important that the optical properties of reference phantoms be measured in a manner that is traceable to the international system of units. SI traceability insures long term consistency of results and will therefore improve the effectiveness of diffuse optics research effort more effective by reducing unwanted variability in the data produced and shared by the community. The ultimate benefit of rigorous SI traceability is the reduction of variability in the data produced by novel diagnostic devices, which will in turn increase the statistical power of clinical trials aiming at validating their clinical usefulness. SI traceability, and therefore uncertainty analysis, is also relevant to traceability aspects mandated by FDA regulations. SI traceability is achieved through a thorough analysis of the measurement principle and its potential error sources. The uncertainty analysis should be ultimately validated by inter-laboratory comparison until a consensus is attained on the best practices for measuring the optical properties of tissue phantoms.

There is a pressing need for a passive optical fiber dosimeter probe for use in real-time monitoring of radiation dose delivered to clinical radiation therapy patients. An optical fiber probe using radiochromic material has been designed and fabricated based on a thin film of the radiochromic material on a dielectric mirror. Measurements of the net optical density vs. time before, during, and after irradiation at a rate of 500cGy/minute to a total dose of 5 Gy were performed. Net optical densities increased from 0.2 to 2.0 for radiochromic thin film thicknesses of 2 to 20 μm, respectively.

We fabricated permanent solid polyurethane-based phantoms in which fluorophores were homogeneously incorporated. For this study, fluorophores of three different families were used: Cyanines, Alexa Fluor and Quantum Dots. The goal of this study was to evaluate the impact of casting the fluorophores in a polyurethane matrix on their optical properties, more specifically the absorbance, molecular extinction coefficient, emission of fluorescence and the resultant fluorescence intensity. All measurements were carried out with 5 concentrations of each fluorophores embedded in polyurethane and in solution. Stability over time was also monitored for a three months period. The casting of fluorophores affects the optical properties of the three dyes under study. The max absorbance, the fluorescence emission and intensity along with the molar extinction coefficient were all affected. Quantum dots behave differently to the cyanine and Alexa Fluor dyes. It was also observed that the incorporation of dyes enables long-term stability of the fluorescence signal.

An endoscope capable of Coherent Anti-Stokes Raman scattering (CARS) imaging would be of significant clinical value for improving early detection of endoluminal cancers. However, developing this technology is challenging for many reasons. First, nonlinear imaging techniques such as CARS are single point measurements thus requiring fast scanning in a small footprint if video rate is to be achieved. Moreover, the intrinsic nonlinearity of this modality imposes several technical constraints and limitations, mainly related to pulse and beam distortions that occur within the optical fiber and the focusing objective. Here, we describe the design and report modeling results of a new CARS endoscope. The miniature microscope objective design and its anticipated performance are presented, along with its compatibility with a new spiral scanningfiber imaging technology developed at the University of Washington. This technology has ideal attributes for clinical use, with its small footprint, adjustable field-of-view and high spatial-resolution. This compact hybrid fiber-based endoscopic CARS imaging design is anticipated to have a wide clinical applicability.

The detection, identification and quantification of pathogenic microorganisms at low cost are of great interest to the agro-food industry. We have developed a simple, rapid, sensitive, and specific method for detection of food-borne pathogens based on use of nanoparticles alongside a low cost fluorescence cell reader for the bioassay. The nanoparticles are coupled with antibodies that allow specific recognition of the targeted Listeria in either a liquid or food matrix. The bioconjugated nanoparticles (FNP) contain thousands of dye molecules enabling significant amplification of the fluorescent signal emitted from each bacterium. The developed fluorescence Cell Reader is an LED-based reader coupled with suitable optics and a camera that acquires high resolution images. The dedicated algorithm allowed the counting of each individual nanoparticles-fluorescent bacterial cells thus enabling highly sensitive reading. The system allows, within 1 hour, the recovery and counting of 10 4 to 10 8 cfu/mL of Listeria in pure culture. However, neither the Cell Reader nor the algorithm can differentiate between the FNPs specifically-bound to the target and the residual unbound FNPs limiting sensitivity of the system. Since FNPs are too small to be washed in the bioassay, a dual tagging approach was implemented to allow online optical separation of the fluorescent background caused by free FNPs.

Solid tissue phantom are the preferred tool for the development, validation, testing and calibration of photon migration instrument. Accuracy, or trueness, of the optical properties of reference phantoms is of the utmost importance as they will be used as the conventional true value against which instrument errors will be evaluated. A detailed quantitative analysis of the uncertainty of time-resolved transmittance characterization of solid optical tissue phantom is presented. Random error sources taken into account are Poisson noise of the photon counting process, additive dark count noise and instrument response function stability. Systematic error sources taken into account are: phantom thickness uncertainty, refractive index uncertainty, time correlated single photon counting system time base calibration uncertainty. Correction procedures for these systematic errors are presented whenever a correction is possible.

Accuracy of time resolved transmittance characterization of solid tissue phantoms is investigated by measuring samples of various geometries with non negligible boundary effects. Relative invariance to geometry provides confidence on absolute accuracy of characterization.

The effect of a magnetic field on the steady-state and time-resolved optical emission of a custom fullerene-linked photosensitizer (PS) in liposome cell phantoms was studied at various oxygen concentrations (0.19-190 microM). Zeeman splitting of the triplet state and hyperfine coupling, which control intersystem crossing between singlet and triplet states, are altered in the presence of low magnetic fields (B320 mT), perturbing the luminescence intensity and lifetime as compared to the triplet state at B = 0. Measurements of the luminescence intensity and lifetime were performed using a time-domain apparatus integrated with a magnet. We propose that by probing magnet-affected optical emissions, one can monitor the state of oxygenation throughout the course of photodynamic therapy. Since the magnetic field effect (MFE) operates primarily by affecting the radical ion pairs related to type I photodynamic action, the enhancement or suppression of the MFE can be used as a measure of the dynamic equilibrium between the type I and II photodynamic pathways. The unique photo-initiated charge-transfer properties of the PS used in this study allow it to serve as both cytotoxic agent and oxygen probe that can provide in situ dosimetric information at close to real time.

The time-resolved fluorescence of photosensitizers (PSs) of varying hydrophobicities, di-and tetrasulfonated Al phthalocyanines (Al-2 and Al-4), and Photochlor (HPPH), was investigated in liposomes used as cell-mimetic models. Using frequency-and time-domain apparatus, the fluorescence lifetime, tau(fluo), was compared for PSs free in aqueous solution and in a liposome-associated state at varied temperatures (25 to 78 degrees C) and oxygen concentrations (0-190 microM). The analysis of tau(fluo) revealed different decay behaviors for the free-solution and liposome-confined PSs, most significantly for the lipophilic HPPH. Hydrophilic PS drugs (Al-4, Al-2) were less affected by the liposomal confinement, depending on the relative hydrophilicity of the compound and the consequent localization in liposomes. Changes in the emission decay due to confinement were detected as differences in the lifetime between the bulk solution and the liposome-localized PS in response to heating and deoxygenation. Specifically, hydrophilic Al-4 produced an identical lifetime trend as a function of temperature both in solu and in a liposome-confined state. Hydrophobic HPPH exhibited a fundamental transformation in its fluorescence decay kinetics, transitioning from a multiexponential (in free solution) to single-exponential (in liposome) decay. Deoxygenation resulted in a ubiquitous tau(fluo) increase for all PSs in free solution, while the opposite, a tau(fluo) decrease, occurred in all liposomal PSs.

A broadband frequency domain fluorescence lifetime system (from ns to ms time scale) has been developed to study the photochemical and photodynamic behavior of model, well-controlled photosensitizer-encapsulating liposomes. Liposomes are known to be efficient and selective photosensitizer (PS) drug delivery vesicles, however, their chemical and physical effects on the photochemical properties of the photosensitizer have not been well characterized. The liposomes employed in this study (both blank and photosensitizer-complexed) were characterized to determine their: a) size distribution (dynamic light scattering), b) image (scanning electron microscope, confocal fluorescence microscopy), c) concentration of particles (flow cytometry), d) temperature-dependant phase transition behavior (differential scanning calorimetry, and e) spectrofluorescent spectrophotometric properties, e.g. aggregation, in the confined environment. The fluorescence decay behavior of two families of encapsulated photosensitizers, di-and tetrasulfonated metallophthalocyanines, and 2-(l-hexyloxyethyl)-2-devinyl pyropheophorbide (HPPH), has been examined as a function of the liposome’s physical properties (size-scale, distribution and concentration of scatterer) and the impact of the photosensitizer spatial confinement determined. It is found that the achievable size range and distribution of the PS- liposomes is controlled by the chemical nature of the PS for large liposomes (1000 nm), and is PS independent for small PS-liposomes (~140nm). The lifetime decay behavior was studied for all three photosensitizer-liposome systems and compared before and after confinement. We found the nature of the decay to be similar before and after encapsulation for the sulfonated phthalocyanines containing ionic moieties (primarily monoexponential) but not for HPPH. In the latter, the decay transitioned from multi- to monoexponential decay upon localizing lypophilic HPPH to the liposomal membrane. This behavior was confirmed by obtaining a similar change in lifetime response with an independent timedomain system. We also varied the environment in temperature and oxygen content to examine the effects on the fluorescent lifetimes of the liposomal complexes. The fluorescence decay of all three PS-containing liposomes showed that the local spatial confinement of PS (dictated by the PS chemistry) into different domains within the liposome directly controls the temperature-response. Membrane-bound photosensitizers were less sensitive to temperature effects as illustrated by the decay dynamics observed in solu, that is, they developed a unique decay behavior that correlated with the phase transition of the membrane. The fluorescent lifetime of PS-encapsulated liposomes in deoxygenated environments, relevant to oxygen independent type I phototoxicity, was also probed in the frequency-domain revealing that liposome-confined PS display very different trends than those observed in solu.

The adsorption of a 14-amino acid amphiphilic peptide, LK14, which is composed of leucine (L, nonpolar) and lysine (K, charged), on hydrophobic polystyrene (PS) and hydrophilic silica (SiO2) was investigated in situ by quartz crystal microbalance (QCM), atomic force microscopy (AFM), and sum frequency generation (SFG) vibrational spectroscopy. The LK14 peptide, adsorbed from a pH 7.4 phosphate-buffered saline (PBS) solution, displayed very different coverage, surface roughness and friction, topography, and surface-induced orientation when adsorbed onto PS versus SiO2 surfaces. Real-time QCM adsorption data revealed that the peptide adsorbed onto hydrophobic PS through a fast (t2 min) process, while a much slower (t30 min) multistep adsorption and rearrangement occurred on the hydrophilic SiO2. AFM measurements showed different surface morphologies and friction coefficients for LK14 adsorbed on the two surfaces. Surface-specific SFG spectra indicate very different ordering of the adsorbed peptide on hydrophobic PS as compared to hydrophilic SiO2. At the LK14 solution/PS interface, CH resonances corresponding to the hydrophobic leucine side chains are evident. Conversely, only NH modes are observed at the peptide solution/SiO2 interface, indicating a different average molecular orientation on this hydrophilic surface. The surface-dependent difference in the molecular-scale peptide interaction at the solution/hydrophobic solid versus solution/hydrophilic solid interfaces (measured by SFG) is manifested as significantly different macromolecular-level adsorption properties on the two surfaces (determined via AFM and QCM experiments).

The kinetics of electrostatic layer-by-layer adsorption of a weakly charged polycation, poly(allylamine hydrochloride), PAH, and a polyanion containing an azobenzene chromophore, P-Azo, was studied using UV-vis spectroscopy and ellipsometry. The thickness of the multilayer films was first measured over the adsorption pH range of 3 to 11, and the growth of multilayers was examined as function of time and concentration. Films assembled in bath pH near that of their pKa value produced both the thickest films, and displayed remarkably rapid adsorption isotherms. In some PAH/P-Azo films a significantly large thickness was achieved in less than 5 seconds, which is more than two orders of magnitude faster than what is usually observed. We show that this anomalously rapid adsorption is a consequence of the weak acid-base nature of the layers. Self-assembled polymer films containg azobenzene groups are interesting materials as the chromophores can be addressed as light-responsive groups for surface patterning and sensing.

Dental implantology procedures necessitate surgical drilling of a hole in the jawbone to insert an artificial root over which a dental prosthesis is placed. The success factor of a dental implant is attaining successful osseointegration. This implies a deeper placed implant closest to the Inferior Alveolar Nerve (IAN) but not too close that it carries the risk of damaging this nerve-artery bundle located along the mandible line. Implications for perforating the IAN in patients can vary from temporary numbness to permanent loss of sensation in the lower facial area. Surgeons routinely use computer-assisted navigation techniques relying on static preoperative cone-beam X-ray computed tomography or other imaging methods such as Magnetic Resonance Imaging (MRI) to assess optimal anatomical distances in the patient’s jaw. However, variability of the measured IAN bundle position with these techniques is at the moment accurate to 30% at best. These techniques are known to introduce localization errors and do not benefit from in situ guidance. Therefore there is an important need for an online, in vivo probe to assess drill distances to a critical nerve-artery bundle in situ to not only avoid jaw paralysis but to improve overall outcome of the implantation. To this aim, we investigate a combined multimodal approach using Optical Coherence Tomography (OCT) for high resolution structural imaging, and functional NIR absorption detection in an optical fiber probe eventually small enough to fit into a surgical drill bit, for real-time evaluation of the distance from the probe to the IAN. We designed a reflectance fiber-optic probe to sense the pulsation of the artery (Heartbeat ~1-2Hz) in the IAN bundle, and a detection circuit board with a high gain (2 . 106 V/A), and very low noise (10mVRMS at output) and bandwidth (10Hz) shown on Figure 1. The success of this NIR channel for accurate real-time proximity sensing by capturing low frequency pulsing signals relies on an ultra low noise photon detection module. Measurements with this fiber probe were conducted at probe-target distances varying from 0mm (probe in contact) to 3mm. The results obtained are promising: the oscillating RMS amplitude varies as a function of distance in a similar fashion to previously published work in pulse oximetry literature [1]. The DC level also has a specific trend as a function of distance. Both AC and DC signal components can be used to extract useful distance information. Original designs using a lock-in amplifier to measure the very low RMS amplitude of the of the back-reflected light intensity, modulated by the spatially oscillating absorption changes due to flowing blood surrogate in an artery simulating tube were promising, but limited. Lock-in signal detection with a reference signal modulated at only 1-2 Hz needs filtering with long time constants. Moreover, clinical implementation with the heartbeat as the reference signal would make real-time measurement impractical. As such, we have developed a flexible optoelectronic detection platform with tunable bandwidth, gain and bias enabling the system to not be limited by electronics noise. Using custom phantoms of the jawbone and including a surrogate arterial dynamic pumping circuit, we demonstrated proof of concept for potential detection range of 0.5-4 mm at l-850nm with a source-detector separation of 1.8mm using the pulsating signal from an artery simulating tube. In compliment, a swept-source OCT at 1.3mm provided finer resolution sensitivity to the proximity of the IAN bundle in the 0-0.9mm range and offered the possibility of imaging the inhomogeneous IAN interface. Methods for calibrating and processing the data to provide robust long range NIR finding capabilities in combination with short-range high precision OCT imaging towards a complete clinical solution will be discussed.

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Preferential tumor localization and the aggregation state of photosensitizers (PSs) can depend on the hydrophilic/hydrophobic nature of the molecule and affect their phototoxicity. In this study, three PSs of different hydrophilicity are introduced in liposomes to understand the structure-photochemistry relationship of PSs in this cellular model system. Absorbance and fluorescence spectra of amphiphilic aluminum (III) phthalocyanine disulfonate chloride adjacent isomer (Al-2), hydrophilic aluminum (III) phthalocyanine chloride tetrasulfonic acid (Al-4), and lipophilic 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide (HPPH) are compared in a liposomal confined state with free PS in bulk solution. For fluorescence measurements, a broad range of concentrations of both bulk and liposomal confined PSs are examined to track the transition from monomers to dimers or higher order aggregates. Epifluorescence microscopy, absorbance, and fluorescence measurements all confirm different localization of the PSs in liposomes, depending on their hydrophilicity. In turn, the localization affects the aggregation of molecules inside the liposome cell model. Data obtained with such cellular models could be useful in optimizing the photochemical properties of photosensitizing drugs based on their structure-dependent interactions with cellular media and subcellular organelles.

Intrinsic reduced scattering coefficient of Intralipid and intrinsic absorption coefficient of Indian ink at NIR wavelengths are accurately assessed in a multi-center study involving different techniques, instrumental set-ups, and analysis methods.