A one-step rapid and ultrasensitive immunoassay capable of detecting proteins in blood serum is developed using gold nanoprobes and fluorescence correlation spectroscopy (FCS). In this approach we take advantage of the inherent photoluminescence property of gold nanoparticles (GNPs) to develop a fluorophore-free assay to observe binding entities by monitoring the diffusion of bound versus unbound molecules in a limited confocal volume. 40-nm GNPs conjugated separately with rabbit anti-IgG (Fc) and goat anti-IgG (Fab) when incubated in blood serum containing IgG forms a sandwich structure constituting dimers and oligomers that can be differentiated by to detect IgG in blood serum at a limit of detection (LOD) of 5 pg/ml. The novelty of integrating GNPs with FCS to develop a sensitive blood immunoassay brings single molecule methods one step closer to the clinic.

Förster resonance energy transfer (FRET) between quantum dot (QD) donors and red fluorescent protein (RFP)-tagged integrin acceptors in live cells is reported for the first time. A silica microsphere was coated with CdSe/ZnS QDs and mounted to the cantilever of an atomic force microscope (AFM). The QD microsphere is then conjugated with fibronectin to bind with RFP-αv integrins expressed on the surface of HeLa cells. Following AFM-controlled cell contact with the QD-microsphere structure, FRET is observed between the QD-RFP pair using a photobleaching measurement technique. This FRET probe technique provides a novel tool for studying the cell surface receptor-ligand interactions in biomedical and biological research.

Optical-resolution photoacoustic microscopy (OR-PAM) is applied to functional brain imaging in living mice. A near-diffraction-limited bright-field optical illumination is employed to achieve micrometer lateral resolution, and a dual-wavelength measurement is utilized to extract the blood oxygenation information. The variation in hemoglobin oxygen saturation (sO₂) along vascular branching has been imaged in a precapillary arteriolar tree and a postcapillary venular tree, respectively. To the best of our knowledge, this is the first report on in vivo volumetric imaging of brain microvascular morphology and oxygenation down to single capillaries through intact mouse skulls. It is anticipated that: (i) chronic imaging enabled by this minimally invasive procedure will advance the study of cortical plasticity and neurological diseases; (ii) revealing the neuroactivity-dependent changes in hemoglobin concentration and oxygenation will facilitate the understanding of neurovascular coupling at the capillary level; and (iii) combining functional OR-PAM and high-resolution blood flowmetry will have the potential to explore cellular pathways of brain energy metabolism.

Measurement of tissue optical properties impacts both optical diagnostic and theraputic applications. Although existing photoacoustic imaging techniques provide optical absorption contrast, we present a photoacoustic technique that demonstrates sensitivity to the optical scattering coefficient of a turbid medium. By incrementing the distance between a surface illumination spot and a subsurface absorber and measuring the photoacoustic amplitude of the absorber, we can effectively estimate the Green's function of light transport in a turbid medium. Our results for different concentrations of Intralipid indicate that this technique has the ability to distinguish small changes of the reduced scattering coefficient. It has the potential to be used for in vivo studies to obtain reduced scattering coefficients of biological tissues. These findings will potentially improve the calculation of subcutaneous fluence in photoacoustic-based techniques and laser dosimetry studies in live tissues.

Optical microanglography (OMAG) is a recently developed imaging modality capable of volumetric imaging of dynamic blood perfusion, down to capillary level resolution, with an imaging depth up to 2.00 mm beneath the tissue surface. We report the use of OMAG to monitor the cerebral blood flow (CBF) over the cortex of mouse brain upon traumatic brain injury (TBI), with the cranium left intact, for a period of two weeks on the same animal. We show the ability of OMAG to repeatedly image 3-D cerebral vasculatures during pre- and post-traumatic phases, and to visualize the changes of regulated CBF and the vascular plasticity after TBI. The results indicate the potential of OMAG to explore the mechanism involved in the rehabilitation of TBI.

We demonstrate that stimulated parametric emission (SPE) microscopy enables label-free, 3-D visualization of internal hemoglobin distribution of live mouse and chicken erythrocytes with high sensitivity. Change in hemoglobin distribution in chicken erythrocytes before and after ethanol fixation is clearly visualized.

Based on the capability of modulating fluorescence intensity by specific molecular events, we report a new multimodal optical-nuclear molecular probe with complementary reporting strategies. The molecular probe (LS498) consists of tetraazacyclododecanetetraacetic acid (DOTA) for chelating a radionuclide, a near-infrared fluorescent dye, and an efficient quencher dye. The two dyes are separated by a cleavable peptide substrate for caspase-3, a diagnostic enzyme that is upregulated in dying cells. LS498 is radiolabeled with ⁶⁴Cu, a radionuclide used in positron emission tomography. In the native form, LS498 fluorescence is quenched until caspase-3 cleavage of the peptide substrate. Enzyme kinetics assay shows that LS498 is readily cleaved by caspase-3, with excellent enzyme kinetic parameters k_cat and K_M of 0.55±0.01 s^-1 and 1.12±0.06 μM, respectively. In mice, the initial fluorescence of LS498 is ten-fold less than control. Using radiolabeled ⁶⁴Cu-LS498 in a controlled and localized in-vivo model of caspase-3 activation, a time-dependent five-fold NIR fluorescence enhancement is observed, but radioactivity remains identical in caspase-3 positive and negative controls. These results demonstrate the feasibility of using radionuclide imaging for localizing and quantifying the distribution of molecular probes and optical imaging for reporting the functional status of diagnostic enzymes.

The salivary pellicle plays an important role in oral physiology, yet noninvasive in situ characterization and mapping of this layer remains elusive. The goal of this study is to develop an optical approach for the real-time, noninvasive mapping and characterization of salivary pellicles using optical coherence tomography (OCT) and optical coherence microscopy (OCM). The long-term goals are to improve diagnostic capabilities in the oral cavity, gain a better understanding of physiological and pathological processes related to the oral hard tissues, and monitor treatment responses. A salivary pellicle is incubated on small enamel cubes using human whole saliva. OCT and OCM imaging occurs at 0, 10, 30, 60 min, and 24 h. For some imaging, spherical gold nanoparticles (15 nm) are added to determine whether this would increase the optical signal from the pellicle. Multiphoton microscopy (MPM) provides the baseline information. In the saliva-incubated samples, a surface signal from the developing pellicle is visible in OCT images. Pellicle "islands" form, which increase in complexity over time until they merge to form a continuous layer over the enamel surface. Noninvasive, in situ time-based pellicle formation on the enamel surface is visualized and characterized using optical imaging.

Previous studies have demonstrated that polarization-sensitive optical coherence tomography (PS-OCT) can be used to image caries lesions in dentin, measure nondestructively the severity of dentin demineralization, and determine the efficacy of intervention with anticaries agents including fluoride and lasers. The objective of this study is to determine if PS-OCT can be used to nondestructively measure a reduction in the reflectivity of dentin lesions after exposure to a remineralization solution. Although studies have shown the ability of PS-OCT to image the remineralization of lesions in enamel, none have included dentin. PS-OCT images of dentin surfaces are acquired after exposure to an artificial demineralizing solution for six days and a remineralizing solution for 20 days. The integrated reflectivity, depth of demineralization, and thickness of the layer of remineralization are calculated for each of the two treatment groups on each sample. Polarized light microscopy and microradiography are used to measure lesion severity on histological thin sections for comparison. PS-OCT successfully measured the formation of a layer of increased mineral content near the lesion surface. Polorized light microscopy (PLM) and transverse microradiography (TMR) corroborated those results. PS-OCT can be used for the nondestructive measurement of the remineralization of dentin.

The eye is not a centered system. The line of sight connects the fovea with the center of the pupil and is usually tilted in the temporal direction. Thus, off-axis optical aberrations, mainly coma and oblique astigmatism, are introduced at the fovea. Tabernero et al. [J. Opt. Soc. Am. A 24(10), 3274-3283 (2007)] showed that a horizontal tilt of the crystalline lens generates a horizontal coma aberration that is compensated by the oblique light incidence on the eye. Here we suggest that corneal astigmatism may also play a role in compensation of oblique aberrations, and we propose a simple model to analyze such a possibility. A theoretical Kooijman eye model with a slight (~0.6 D) with-the-rule astigmatism is analyzed. Light rays at different incidence angles to the optical axis are considered, and the corresponding point spread functions (PSFs) at the retina are calculated. A quality criterion is used to determine the incidence angle that provides the narrowest and highest PSF energy peak. We show that the best image is obtained for a tilted incidence angle compatible with mean values of the angle kappa. This suggests that angle kappa, lens tilt, and corneal astigmatism may combine to provide a passive compensation mechanism to minimize aberrations on the fovea.

The intrinsic turbidity of scaffolds formed by natural biomaterials such as collagen fibers prevents high-resolution light microscopy in depth. In this research, we have developed a new method of using light microscopy for penetrative three-dimensional (3-D) visualization of scaffolds formed by collagen, chitosan, or cellulose. First, we applied an optical-clearing solution, FocusClear, to permeate and reduce the turbidity of the scaffolds. The improved photon penetration allowed fluorophores for efficient excitation and emission in the FocusClear solution. Confocal microscopy was applied to achieve cellular-level resolution up to 350 µm for both the fibroblast/collagen and the osteoblast/chitosan constructs and micrometer-level resolution up to 40 μm for the cellulose membrane. The depth of imaging of the cellulose membrane was further improved to 80 μm using two-photon microscopy. Significantly, these voxel-based confocal/two-photon micrographs allowed postrecording image processing via Amira projection algorithms for 3-D visualization and analysis of the scanned region. Although this optical method remains limited in viewing block scaffolds in thin sections, our approach provides a noninvasive way to microscopically examine the scaffold structure, which would be a valuable tool to studying biomaterials and their interactions with the molecule/cell of interest within the scaffold in an integrated fashion.

The chest wall underneath the breast tissue affects near-infrared (NIR) diffusive waves measured with reflection geometry. With the assistance of a co-registered ultrasound, the depth and the tilting angle of the chest wall can be determined and are used to model the breast as a two-layer medium. Finite element method (FEM) is suitable for modeling complex boundary conditions and is adapted to model the breast tissue and chest wall. Four parameters of bulk absorption and reduced scattering coefficients of these two layers are estimated and used for imaging reconstruction. Using a two-layer model, we have systematically investigated the effect of the chest wall on breast lesion reconstruction. Results have shown that chest-wall depth, titling angle, and difference between optical properties of two layers of lesion and reference sites affect the lesion reconstruction differently. Our analysis will be valuable and informative to researchers who are using reflectance geometry for breast imaging. The analysis can also provide guidelines for imaging operators to minimize image artifacts and to produce the best reconstruction results.

Fourier transform infrared (FTIR) spectroscopy is sensitive to the molecular composition of tissue and has the potential to identify premalignant tissue (dysplasia) as an adjunct to endoscopy. We demonstrate collection of mid-infrared absorption spectra with a silver halide (AgCl_0.4Br_0.6) optical fiber and use spectral preprocessing to identify optimal subranges that classify colonic mucosa as normal, hyperplasia, or dysplasia. We collected spectra (n=83) in the 950 to 1800 cm^-1 regime on biopsy specimens obtained from human subjects (n=37). Subtle differences in the magnitude of the absorbance peaks at specific wave numbers were observed. The best double binary algorithm for distinguishing normal-versus-dysplasia and hyperplasia-versus-dysplasia was determined from an exhaustive search of spectral intervals and preprocessing techniques. Partial least squares discriminant analysis was used to classify the spectra using a leave-one-subject-out cross-validation strategy. The results were compared with histology reviewed independently by two gastrointestinal pathologists. The optimal thresholds identified resulted in an overall sensitivity, specificity, accuracy, and positive predictive value of 96%, 92%, 93%, and 82%, respectively. These results indicated that mid-infrared absorption spectra collected remotely with an optical fiber can be used to identify colonic dysplasia with high accuracy, suggesting that continued development of this technique for the early detection of cancer is promising.

The hearing performance with conventional hearing aids and cochlear implants is dramatically reduced in noisy environments and for sounds more complex than speech (e. g. music), partially due to the lack of localized sensorineural activation across different frequency regions with these devices. Laser light can be focused in a controlled manner and may provide more localized activation of the inner ear, the cochlea. We sought to assess whether visible light with parameters that could induce an optoacoustic effect (532 nm, 10-ns pulses) would activate the cochlea. Auditory brainstem responses (ABRs) were recorded preoperatively in anesthetized guinea pigs to confirm normal hearing. After opening the bulla, a 50-μm core-diameter optical fiber was positioned in the round window niche and directed toward the basilar membrane. Optically induced ABRs (OABRs), similar in shape to those of acoustic stimulation, were elicited with single pulses. The OABR peaks increased with energy level (0.6 to 23 μJ/pulse) and remained consistent even after 30 minutes of continuous stimulation at 13 μJ, indicating minimal or no stimulation-induced damage within the cochlea. Our findings demonstrate that visible light can effectively and reliably activate the cochlea without any apparent damage. Further studies are in progress to investigate the frequency-specific nature and mechanism of green light cochlear activation.

Diffusion of two model drugs-benzyl nicotinate and ibuprofen-and the plasma macromolecule albumin across atherosclerotic rabbit aorta was studied ex vivo by attenuated total reflection-Fourier transform infrared (ATR-FTIR) imaging. Solutions of these molecules were applied to the endothelial surface of histological sections of the aortic wall that were sandwiched between two impermeable surfaces. An array of spectra, each corresponding to a specific location in the section, was obtained at various times during solute diffusion into the wall and revealed the distribution of the solutes within the tissue. Benzyl nicotinate in Ringer's solution showed higher affinity for atherosclerotic plaque than for apparently healthy tissue. Transmural concentration profiles for albumin demonstrated its permeation across the section and were consistent with a relatively low distribution volume for the macromolecule in the middle of the wall. The ability of albumin to act as a drug carrier for ibuprofen, otherwise undetected within the tissue, was demonstrated by multivariate subtraction image analysis. In conclusion, ATR-FTIR imaging can be used to study transport processes in tissue samples with high spatial and temporal resolution and without the need to label the solutes under study.

Liposomal formulations of drugs have been shown to enhance drug efficacy by prolonging circulation time, increasing local concentration and reducing off-target effects. Controlled release from these formulations would increase their utility, and hyperthermia has been explored as a stimulus for targeted delivery of encapsulated drugs. Use of lasers as a thermal source could provide improved control over the release of the drug from the liposomes with minimal collateral tissue damage. Appropriate methods for assessing local release after systemic delivery would aid in testing and development of better formulations. We use in vivo bioluminescence imaging to investigate the spatiotemporal distribution of luciferin, used as a model small molecule, and demonstrate laser-induced release from liposomes in animal models after systemic delivery. These liposomes were tested for luciferin release between 37 and 45 °C in PBS and serum using bioluminescence measurements. In vivo studies were performed on transgenic reporter mice that express luciferase constitutively throughout the body, thus providing a noninvasive readout for controlled release following systemic delivery. An Nd:YLF laser was used (527 nm) to heat tissues and induce rupture of the intravenously delivered liposomes in target tissues. These data demonstrate laser-mediated control of small molecule delivery using thermally sensitive liposomal formulations.

Texture analysis for tissue characterization is a current area of optical coherence tomography (OCT) research. We discuss some of the differences between OCT systems and the effects those differences have on the resulting images and subsequent image analysis. In addition, as an example, two algorithms for the automatic recognition of bladder cancer are compared: one that was developed on a single system with no consideration for system differences, and one that was developed to address the issues associated with system differences. The first algorithm had a sensitivity of 73% and specificity of 69% when tested using leave-one-out cross-validation on data taken from a single system. When tested on images from another system with a different central wavelength, however, the method classified all images as cancerous regardless of the true pathology. By contrast, with the use of wavelet analysis and the removal of system-dependent features, the second algorithm reported sensitivity and specificity values of 87 and 58%, respectively, when trained on images taken with one imaging system and tested on images taken with another.

A noninvasive methodology, combining functional electrical stimulation and time-domain near-infrared spectroscopy (TD-NIRS), is developed to verify whether stroke-altered muscular metabolism on postacute patients. Seven healthy subjects and nine postacute stroke patients undergo a protocol of knee flex-extension induced by quadricep electrical stimulation. During the protocol, TD-NIRS measurements are performed on both rectus femoris to investigate whether significant differences arise between able-bodied and stroke subjects and between patients' paretic and healthy legs. During baseline, metabolic parameters do not show any significant differences among subjects. During stimulation, paretic limbs produce a knee angle significantly lower than healthy legs. During recovery, patients' healthy limbs show a metabolic behavior correlated to able-bodied subjects. Instead, the correlation between the metabolic behavior of the paretic and able-bodied legs allows the definition of two patients' subgroups: one highly correlated (R>0.87) and the other uncorrelated (R<0.08). This grouping reflects the patient functional condition. The results obtained on the most impaired patients suggest that stroke does not produce any systemic consequences at the muscle, but the metabolic dysfunction seems to be local and unilateral. It is crucial to enlarge the sample size of the two subgroups before making these preliminary results a general finding.

The cornea functions as an optical lens and plays an important role in vision. For corneal diagnosis and treatment such as refractive surgery, a microscopic imaging system with a 3-D cellular resolution and retinal safety is strongly desired. Recently, confocal and multiphoton microscopies have been applied to corneal imaging with visible to near-infrared light sources. To increase retinal safety, an infrared light source is be needed. In this work, an infrared-based third and second harmonic generation microscopic study of mouse eyes is reported with ~700-μm penetrability and high cellular resolution. This study provides a critical reference for future development of infrared-based corneal imaging.

We develop a standardized, fully automated, quantification system for liver fibrosis assessment using second harmonic generation microscopy and a morphology-based quantification algorithm. Liver fibrosis is associated with an abnormal increase in collagen as a result of chronic liver diseases. Histopathological scoring is the most commonly used method for liver fibrosis assessment, where a liver biopsy is stained and scored by experienced pathologists. Due to the intrinsic limited sensitivity and operator-dependent variations, there exist high inter- and intraobserver discrepancies. We validate our quantification system, Fibro-C-Index, with a comprehensive animal study and demonstrate its potential application in clinical diagnosis to reduce inter- and intraobserver discrepancies.

A Hartmann-Shack wavefront sensor (HSWS) has been proven to be a reliable tool for the quantitative analysis of human ocular aberrations. In an active adaptive optics (AO) system, it has the role to monitor wave aberrations. To ensure the exclusive retrieval of Zernike coefficients for the measured ocular wavefronts, we first nullify the AO system's aberrations. This is of particular importance in our setup with a twisted-nematic (TN) liquid-crystal-on-silicon (LCoS) chip as the wavefront manipulator due to its strong unwanted zero-order diffractive beam. We characterize the AO system's performance-before and after ocular corrections-by means of different parameters, including experimental and simulated point spread functions (PSFs). An iterative closed-loop algorithm reduces the residual wavefront error to typical values of 0.1 μm. This system constitutes a wavefront corrector that can possibly be used for high resolution retinal imaging purposes or for visual psychophysical experiments.

A theoretical model is established for dealing with second-harmonic generation (SHG) in type I collagen excited by linearly polarized light focused by a microscope. With this model, the effects of the polarization angle α, numerical aperture (NA), as well as the ratio of hyperpolarizability ρ=β xxx/β xyy on SHG emission have been investigated. Simulation results reveal that SHG emission power changes periodically as α. The use of lower NA leads to weaker SHG emission but is more concentrated in two closer lobes, whereas more distributed emission in two detached lobes appear at higher NA. As the introduction of polarization direction, which is not along with the fiber axis (alpha ≠ 0 deg), one more element β xyy is valid in our case than β xxx alone, while their ratio ρ plays a very important role for collagen features characterization. SHG emission with ρ shows complicated modality that SHG emission is different at different α and not symmetric at ± ρ except at α=0 deg, suggesting the important impact of polarization working on ρ for SHG emission. Our theoretical simulation results provide useful clues for experimental study of microscopic SHG emission in collagen excited by linearly polarized beam.

Near-infrared spectroscopy is used to quantify the subcutaneous adipose tissue thickness (ATT) over five muscle groups (vastus medialis, vastus lateralis, gastrocnemius, ventral forearm and biceps brachii muscle) of healthy volunteers (n=20). The optical lipid signal (OLS) was obtained from the second derivative of broad band attenuation spectra and the lipid absorption peak (λ=930 nm). Ultrasound and MR imaging as well as mechanical calliper readings were taken as reference methods. The data show that the OLS is a good predictor for ATT (<16 mm) with absolute and relative errors of <0.8 mm and <24%, respectively. The optical method compares favourably with calliper reading. The finding of a non-linear relationship of optical signal vs. ultrasound is explained by a theoretical two-layer model based on the diffusion approximation for the transport of photons. The crosstalk between the OLS and tissue hemoglobin concentration changes during an incremental cycling exercise was found to be small, indicating the robustness of OLS. Furthermore, the effect of ATT on spatially-resolved spectroscopy measurements is shown to decrease the calculated muscle hemoglobin concentration and to increase oxygen saturation.

Stress differences via spectral shifts that arise among failed, strained, and undamaged regions of bone can be determined using Raman spectroscopy and double-notch specimens. A double-notch specimen is a model in which the early stages of fracture can be examined. At four-point bending, fracture occurs at one of the notches. Tissue near each notch is representative of bone in a state either directly before or after bone failure. Raman images are acquired among three regions: control, strained (root of unbroken notch), and failed (root of fractured notch). The center of gravities (CGs), a way to monitor wavenumber shifts, of the phosphate v₁ band are calculated. A PO₄^-3 v₁ band shift most likely corresponds to a change in spacing between phosphate cations and anions. This spectral shift is converted into stress values using the dv/dP coefficient, determined by applying known pressures/stresses and measuring the change in position of the PO₄^-3 v₁ band. In comparison to control regions, the residual stress in strained and failed regions is significantly higher (p=0.0425 and p=0.0169, respectively). In strained regions, residual stress is concentrated near the corners of the unbroken notch, whereas in failed regions the high stresses are confined near the edge of the fracture.

Coherent anti-Stokes Raman scattering (CARS) microscopy is used to determine the distribution and concentration of selected compounds in intact human hair. By generating images based on ratiometric CARS contrast, quantitative concentration maps of both water and externally applied d-glycine are produced in the cortex of human hair fibers. Both water and d-glycine are found to homogeneously distribute throughout the cortical regions of the hair. The ability to selectively detect molecular agents in hair fibers is of direct relevance to understanding the chemical and physical mechanisms that underlie the performance of hair-care products.

Four-dimensional (4-D) imaging of the embryonic heart allows study of cardiac morphology and function in vivo during development. However, 4-D imaging of the embryonic heart using current techniques, including optical coherence tomography (OCT), is limited by the rate of image acquisition. Here, we present a nongated 4-D imaging strategy combined with an efficient postacquisition synchronization procedure that circumvents limitations on acquisition rate. The 4-D imaging strategy acquires a time series of images in B mode at several different locations along the heart, rendering out-of-phase image sequences. Then, our synchronization procedure uses similarity of local structures to find the phase shift between neighboring image sequences, employing M-mode images (extracted from the acquired B-mode images) to achieve computational efficiency. Furthermore, our procedure corrects the phase shifts by considering the phase lags introduced by peristaltic-like contractions of the embryonic heart wall. We applied the 4-D imaging strategy and synchronization procedure to reconstruct the cardiac outflow tract (OFT) of a chick embryo, imaged with OCT at early stages of development (Hamburger-Hamilton stage 18). We showed that the proposed synchronization procedure achieves efficiency without sacrificing accuracy and that the reconstructed 4-D images properly captured the dynamics of the OFT wall motion.

It has long been speculated that underlying variations in tissue anatomy affect in vivo spectroscopic measurements. We investigate the effects of cervical anatomy on reflectance and fluorescence spectroscopy to guide the development of a diagnostic algorithm for identifying high-grade squamous intraepithelial lesions (HSILs) free of the confounding effects of anatomy. We use spectroscopy in both contact probe and imaging modes to study patients undergoing either colposcopy or treatment for HSIL. Physical models of light propagation in tissue are used to extract parameters related to tissue morphology and biochemistry. Our results show that the transformation zone, the area in which the vast majority of HSILs are found, is spectroscopically distinct from the adjacent squamous mucosa, and that these anatomical differences can directly influence spectroscopic diagnostic parameters. Specifically, we demonstrate that performance of diagnostic algorithms for identifying HSILs is artificially enhanced when clinically normal squamous sites are included in the statistical analysis of the spectroscopic data. We conclude that underlying differences in tissue anatomy can have a confounding effect on diagnostic spectroscopic parameters and that the common practice of including clinically normal squamous sites in cervical spectroscopy results in artificially improved performance in distinguishing HSILs from clinically suspicious non-HSILs.

Elastic scattering spectroscopy (ESS) may be used to detect high-grade dysplasia (HGD) or cancer in Barrett's esophagus (BE). When spectra are measured in vivo by a hand-held optical probe, variability among replicated spectra from the same site can hinder the development of a diagnostic model for cancer risk. An experiment was carried out on excised tissue to investigate how two potential sources of this variability, pressure and angle, influence spectral variability, and the results were compared with the variations observed in spectra collected in vivo from patients with Barrett's esophagus. A statistical method called error removal by orthogonal subtraction (EROS) was applied to model and remove this measurement variability, which accounted for 96.6% of the variation in the spectra, from the in vivo data. Its removal allowed the construction of a diagnostic model with specificity improved from 67% to 82% (with sensitivity fixed at 90%). The improvement was maintained in predictions on an independent in vivo data set. EROS works well as an effective pretreatment for Barrett's in vivo data by identifying measurement variability and ameliorating its effect. The procedure reduces the complexity and increases the accuracy and interpretability of the model for classification and detection of cancer risk in Barrett's esophagus.

Our goal is to compare and contrast various image reconstruction algorithms for optoacoustic tomography (OAT) assuming a finite linear aperture of the kind that arises when using a linear-array transducer. Because such transducers generally have tall, narrow elements, they are essentially insensitive to out-of-plane acoustic waves, and the usually 3-D OAT problem reduces to a 2-D problem. Algorithms developed for the 3-D problem may not perform optimally in 2-D. We have implemented and evaluated a number of previously described OAT algorithms, including an exact (in 3-D) Fourier-based algorithm and a synthetic-aperture-based algorithm. We have also implemented a 2-D algorithm developed by Norton for reflection mode tomography that has not, to the best of our knowledge, been applied to OAT before. Our simulation studies of resolution, contrast, noise properties, and signal detectability measures suggest that Norton's approach-based algorithm has the best contrast, resolution, and signal detectability.

We characterize and compare the axial and lateral performance of fluorescence confocal systems imaging in turbid media. The aperture configurations studied are a single pinhole, a slit, a Nipkow disk, and a linear array of pinholes. Systems with parallelized apertures are used clinically because they enable high-speed and real-time imaging. Understanding how they perform in highly scattering tissue is important. A Monte Carlo model was developed to characterize parallelized system performance in a scattering media representative of human tissues. The results indicate that a slit aperture has degraded performance, both laterally and axially. In contrast, the analysis reveals that multipinhole apertures such as a Nipkow disk or a linear pinhole array can achieve performance nearly equivalent to a single pinhole aperture. The optimal aperture spacing for the multipinhole apertures was determined for a specific tissue model. In addition to comparing aperture configurations, the effects of tissue nonradiative absorption, scattering anisotropy, and fluorophore concentration on lateral and axial performance of confocal systems were studied.

The photothermoacoustic (PTA) or photoacoustic (PA) effect induced in light-absorbing materials can be observed either as a transient signal in time domain or as a periodic response to modulated optical excitation. Both techniques can be utilized for creating an image of subsurface light-absorbing structures (chromophores). In biological materials, the optical contrast information can be related to physiological activity and chemical composition of a test specimen. The present study compares experimentally the two PA imaging modalities with respect to the maximum imaging depth achieved in scattering media with optical properties similar to biological tissues. Depth profilometric measurements were carried out using a dual-mode laser system and a set of aqueous light-scattering solutions mimicking photon propagation in tissue. Various detection schemes and signal processing methods were tested to characterize the depth sensitivity of PA measurements. The obtained results demonstrate the capabilities of both techniques and can be used in specific PTA imaging applications for development of image reconstruction algorithms aimed at maximizing system performance. Our results demonstrate that submillimeter-resolution depth-selective PA imaging can be achieved without nanosecond-pulsed laser systems by appropriate modulation of a continuous laser source and a signal processing algorithm adapted to specific parameters of the PA response.

We investigate the possibility of using ALA-derived PpIX fluorescence spectroscopy for the detection of epithelial hyperkeratosis (EH) or epithelial dysplasia (ED) lesions in oral submucous fibrosis (OSF) patients that could not be found by autofluorescence spectroscopy. Twenty percent of ALA solution gel was applied onto oral neoplasia and surrounding normal tissue [normal oral mucosa (NOM)] for 90 min. Fluorescence emission spectra were measured under 410 nm excitation. Generally, the most intense fluorescence emission peaks occurred at 460 and 630 nm. The ratios of the area under red peak (630±10 nm) to the area under blue peak (460±10 nm), denoted as R/B, were calculated. We found that OSF mucosa has the lowest R/B value, followed by NOM, EH on OSF, and ED on OSF. An ANOVA test showed significant differences between OSF, NOM, EH on OSF, and ED on OSF (p<0.05). However, measurements of autofluorescence (i.e., before ALA application) show no significant differences between OSF, NOM, EH on OSF, and ED on OSF (ANOVA test, p>0.05). These results indicate that ALA-induced PpIX fluorescence spectroscopy could be used to identify the premalignant lesions on oral fibrotic mucosa, which could not be found by autofluorescence.

Hemoglobin-based oxygen carriers (HBOCs) are solutions of cell-free hemoglobin (Hb) that have been developed for replacement or augmentation of blood transfusion. It is important to monitor in vivo tissue hemoglobin content, total tissue hemoglobin [THb], oxy- and deoxy-hemoglobin concentrations ([OHb], [RHb]), and tissue oxygen saturation (S_tO₂=[OHb]/[THb]×100%) to evaluate effectiveness of HBOC transfusion. We designed and constructed a broadband diffuse optical spectroscopy (DOS) prototype system to measure bulk tissue absorption and scattering spectra between 650 and 1000 nm capable of accurately determining these tissue hemoglobin component concentrations in vivo. Our purpose was to assess the feasibility of using DOS to optically monitor tissue [OHb], [RHb], S_tO₂, and total tissue hemoglobin concentration ([THb]=[OHb]+[RHb]) during HBOC infusion using a rabbit hypovolemic shock model. The DOS prototype probe was placed on the shaved inner thigh muscle of the hind leg to assess concentrations of [OHb], [RHb], [THb], as well as S_tO₂. Hemorrhagic shock was induced in intubated New Zealand white rabbits (N=6) by withdrawing blood via a femoral arterial line to 20% blood loss (10-15 cc/kg). Hemoglobin glutamer-200 (Hb-200) 1:1 volume resuscitation was administered following the hemorrhage. These values were compared against traditional invasive measurements, serum hemoglobin concentration (sHGB), systemic blood pressure, heart rate, and blood gases. DOS revealed increases of [THb], [OHb], and tissue hemoglobin oxygen saturation after Hb-200 infusion, while blood total hemoglobin values continued did not increase; we speculate, due to hyperosmolality induced hemodilution. DOS enables noninvasive in vivo monitoring of tissue hemoglobin and oxygenation parameters during shock and volume expansion with HBOC and potentially enables the assessment of efficacy of resuscitation efforts using artificial blood substitutes.

A swept-source optical coherence tomography (SS-OCT) system is used to clinically scan oral lesions in different oral carcinogenesis stages, including normal oral mucosa control, mild dysplasia (MiD), moderate dysplasia (MoD), early-stage squamous cell carcinoma (ES-SCC), and well-developed SCC (WD-SCC), for diagnosis purpose. On the basis of the analyses of the SS-OCT images, the stages of dysplasia (MiD and MoD), and SCC (ES-SCC and WD-SCC) can be differentiated from normal control by evaluating the depth-dependent standard deviation (SD) values of lateral variations. In the dysplasia stage, the boundary between the epithelium (EP) and lamina propria (LP) layers can still be identified and the EP layer becomes significantly thicker than that of normal control. Also, in a certain range of the EP layer above the EP/LP boundary, the SD value becomes larger than a certain percentage of the maximum level, which is observed around the EP/LP boundary. On the other hand, in the ES-SCC and WD-SCC stages, the EP/LP boundary disappears. Because of the higher density of connective tissue papillae in the ES-SCC stage, the SD values of the slowly varying lateral scan profiles in the ES-SCC samples are significantly larger than those in the WD-SCC sample. Also, ES-SCC can be differentiated from WD-SCC by comparing the exponential decay constants of averaged A-mode scan profiles. Because of the higher tissue absorption in the WD-SCC lesion, the decay constants in the WD-SCC samples are significantly higher than those in the ES-SCC samples.

Cardiac architecture is inherently three-dimensional, yet most characterizations rely on two-dimensional histological slices or dissociated cells, which remove the native geometry of the heart. We previously developed a method for labeling intact heart sections without dissociation and imaging large volumes while preserving their three-dimensional structure. We further refine this method to permit quantitative analysis of imaged sections. After data acquisition, these sections are assembled using image-processing tools, and qualitative and quantitative information is extracted. By examining the reconstructed cardiac blocks, one can observe end-to-end adjacent cardiac myocytes (cardiac strands) changing cross-sectional geometries, merging and separating from other strands. Quantitatively, representative cross-sectional areas typically used for determining hypertrophy omit the three-dimensional component; we show that taking orientation into account can significantly alter the analysis. Using fast-Fourier transform analysis, we analyze the gross organization of cardiac strands in three dimensions. By characterizing cardiac structure in three dimensions, we are able to determine that the α crystallin mutation leads to hypertrophy with cross-sectional area increases, but not necessarily via changes in fiber orientation distribution.

Successful treatment of cancer is highly dependent on the stage at which it is diagnosed. Early diagnosis, when the disease is still localized at its origin, results in very high cure rates-even for cancers that typically have poor prognosis. Biopsies are often used for diagnosis of disease. However, because biopsies are destructive, only a limited number can be taken. This leads to reduced sensitivity for detection due to sampling error. A real-time fluorescence confocal microlaparoscope has been developed that provides instant in vivo cellular images, comparable to those provided by histology, through a nondestructive procedure. The device includes an integrated contrast agent delivery mechanism and a computerized depth scan system. The instrument uses a fiber bundle to relay the image plane of a slit-scan confocal microlaparoscope into tissue. It has a 3-μm lateral resolution and a 25-μm axial resolution. Initial in vivo clinical testing using the device to image human ovaries has been done in 21 patients. Results indicate that the device can successfully image organs in vivo without complications. Results with excised tissue demonstrate that the instrument can resolve sufficient cellular detail to visualize the cellular changes associated with the onset of cancer.

The cell nucleus is the dominant optical scatterer in the cell. Neoplastic cells are characterized by cell nucleus polymorphism and polychromism-i.e., the nuclei exhibits an increase in the distribution of both size and refractive index. The relative size parameter, and its distribution, is proportional to the product of the nucleus size and its relative refractive index and is a useful discriminant between normal and abnormal (cancerous) cells. We demonstrate a recently introduced holographic technique, digital Fourier microscopy (DFM), to provide a sensitive measure of this relative size parameter. Fourier holograms were recorded and optical scatter of individual scatterers were extracted and modeled with Mie theory to determine the relative size parameter. The relative size parameter of individual melanocyte cell nuclei were found to be 16.5±0.2, which gives a cell nucleus refractive index of 1.38±0.01 and is in good agreement with previously reported data. The relative size parameters of individual malignant melanocyte cell nuclei are expected to be greater than 16.5.

The dermal degeneration accompanying photoaging is considered to promote skin roughness features such as wrinkles. Our previous study demonstrated that polarization-sensitive spectral domain optical coherence tomography (PS-SD-OCT) enabled noninvasive three-dimensional evaluation of the dermal degeneration of photoaged skin as a change in dermal birefringence, mainly due to collagenous structures. Our purpose is to examine the relationship between dermal birefringence and elasticity and the skin morphology in the eye corner area using PS-SD-OCT. Nineteen healthy male subjects in their seventees were recruited as subjects. A transverse dermal birefringence map, automatically produced by the algorithm, did not show localized changes in the dermal birefringence in the part of the main horizontal wrinkle. The averaged upper dermal birefringence, however, showed depth-dependent correlation with the parameters of skin roughness significantly, suggesting that solar elastosis is a major factor for the progress of wrinkles. Age-dependent parameters of skin elasticity measured with Cutometer did not correlate with the parameters. These results suggest that the analysis of dermal birefringence using PS-SD-OCT enables the evaluation of photoaging-dependent upper dermal degeneration related to the change of skin roughness.

The cavernous nerves course along the surface of the prostate and are responsible for erectile function. Improvements in identification, imaging, and visualization of the cavernous nerves during prostate cancer surgery may improve nerve preservation and postoperative sexual potency. Two-dimensional (2-D) optical coherence tomography (OCT) images of the rat prostate were segmented to differentiate the cavernous nerves from the prostate gland. To detect these nerves, three image features were employed: Gabor filter, Daubechies wavelet, and Laws filter. The Gabor feature was applied with different standard deviations in the x and y directions. In the Daubechies wavelet feature, an 8-tap Daubechies orthonormal wavelet was implemented, and the low-pass sub-band was chosen as the filtered image. Last, Laws feature extraction was applied to the images. The features were segmented using a nearest-neighbor classifier. N-ary morphological postprocessing was used to remove small voids. The cavernous nerves were differentiated from the prostate gland with a segmentation error rate of only 0.058±0.019. This algorithm may be useful for implementation in clinical endoscopic OCT systems currently being studied for potential intraoperative diagnostic use in laparoscopic and robotic nerve-sparing prostate cancer surgery.

We utilize multiphoton microscopy for the label-free diagnosis of noncancerous, lung adenocarcinoma (LAC), and lung squamous cell carcinoma (SCC) tissues from humans. Our results show that the combination of second-harmonic generation (SHG) and multiphoton excited autofluorescence (MAF) signals may be used to acquire morphological and quantitative information in discriminating cancerous from noncancerous lung tissues. Specifically, noncancerous lung tissues are largely fibrotic in structure, while cancerous specimens are composed primarily of tumor masses. Quantitative ratiometric analysis using MAF to SHG index (MAFSI) shows that the average MAFSI for noncancerous and LAC lung tissue pairs are 0.55±0.23 and 0.87±0.15, respectively. In comparison, the MAFSIs for the noncancerous and SCC tissue pairs are 0.50±0.12 and 0.72±0.13, respectively. Our study shows that nonlinear optical microscopy can assist in differentiating and diagnosing pulmonary cancer from noncancerous tissues.

A novel method to distribute proteins on solid surfaces is proposed. Proteins microencapsulated in the water pool of reverse micelles were used to coat a solid surface with well-individualized round spots of 1 to 3 μm in diameter. The number of spots per unit area can be increased through the concentration of reverse micelles, and networks of spots were obtained at high concentrations of large reverse micelles. Moreover, depending on the pool size of the water reverse micelles, proteins can be deposited far from each other or in close proximity within the range of 50 to 70 Å. This proximity obtained with small reverse micelles was proved through fluorescence lifetime imaging microscopy and fluorescence resonance energy transfer (FLIM-FRET) measurements for the most relevant FRET pair in cell biology studies, the cyan and yellow fluorescent proteins. This novel procedure has several advantages and reveals the potential for study of protein-protein interactions on solid surfaces and for developing novel biomaterials and molecular devices based on biorecognition elements.

We report the technical feasibility of autofluorescence ductoscopy in the ex-vivo setting. The current imaging algorithm for visualizing tumor tissue against the normal tissue background, although developed and optimized for other organs, appears to provide discrimination between intraductal tumor and normal ductal tissue. Point fluoroscopy is also performed. Although the optical "geometry" for this is different, the findings are consistent with the imaging observations.

Optical coherence tomography (OCT) is a noninvasive, high-resolution imaging technology capable of delivering real-time, near-histologic images of tissues. Mustard gas is a vesicant-blistering agent that can cause severe and lethal damage to airway and lungs. The ability to detect and assess airway injury in the clinical setting of mustard exposure is currently limited. The purpose of this study is to assess the ability to detect and monitor progression of half-mustard [2-chloroethylethylsulfide (CEES)] airway injuries with OCT techniques. A ventilated rabbit mustard exposure airway injury model is developed. A flexible fiber optic OCT probe is introduced into the distal trachea to image airway epithelium and mucosa in vivo. Progression of airway injury is observed over eight hours with OCT using a prototype time-domain superluminescent diode OCT system. OCT tracheal images from CEES exposed animals are compared to control rabbits for airway mucosal thickening and other changes. OCT detects the early occurrence and progression of dramatic changes in the experimental group after exposure to CEES. Histology and immunofluorescence staining confirms this finding. OCT has the potential to be a high resolution imaging modality capable of detecting, assessing, and monitoring treatment for airway injury following mustard vesicant agent exposures.

The relationship between measurements of cerebral blood oxygenation and neuronal activity is highly complex and depends on both neurovascular and neurometabolic biological coupling. While measurements of blood oxygenation changes via optical and MRI techniques have been developed to map functional brain activity, there is evidence that the specific characteristics of these signals are sensitive to the underlying vascular physiology and structure of the brain. Since baseline blood flow and oxygen saturation may vary between sessions and across subjects, functional blood oxygenation changes may be a less reliable indicator of brain activity in comparison to blood flow and metabolic changes. In this work, we use a biomechanical model to examine the relationships between neural, vascular, metabolic, and hemodynamic responses to parametric whisker stimulation under both normal and hypercapnic conditions in a rat model. We find that the relationship between neural activity and oxy- and deoxyhemoglobin changes is sensitive to hypercapnia-induced changes in baseline cerebral blood flow. In contrast, the underlying relationships between evoked neural activity, blood flow, and model-estimated oxygen metabolism changes are unchanged by the hypercapnic challenge. We conclude that evoked changes in blood flow and cerebral oxygen metabolism are more closely associated with underlying evoked neuronal responses.

Recaldent is a product of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP). The remineralizing potential of CPP-ACP per se, or when combined with 0.22% Fl gel on artificially demineralized enamel using laser florescence, is investigated. Mesial surfaces of 15 sound human molars are tested using a He-Cd laser beam at 441.5 nm with 18-mW power as an excitation source on a suitable setup based on a Spex 750-M monochromator provided with a photomultiplier tube (PMT) for detection of collected autofluorescence from sound enamel. Mesial surfaces are subjected to demineralization for ten days. The spectra from demineralized enamel are measured. Teeth are divided into three groups according to the remineralizing regimen: group 1 Recaldent per se, group 2 Recaldent combined with fluoride gel and ACP, and group 3 artificial saliva as a positive control. After following these protocols for three weeks, the spectra from the remineralized enamel are measured. The spectra of enamel autofluorescence are recorded and normalized to peak intensity at about 540 nm to compare spectra from sound, demineralized, and remineralized enamel surfaces. A slight red shift occurred in spectra from demineralized enamel, while a blue shift may occur in remineralized enamel. Group 2 shows the highest remineralizing potential. Combining fluoride and ACP with CPP-ACP can give a synergistic effect on enamel remineralization.

An electrode with adjacent optical fibers for measurements during navigation and radio frequency lesioning in the brain is modeled for Monte Carlo simulations of light transport in brain tissue. Relative reflected light intensity at 780 nm, I₇₈₀, from this electrode and probes with identical fiber configuration are simulated using the intensity from native white matter as reference. Models are made of homogeneous native and coagulated gray, thalamus, and white matter as well as blood. Dual layer models, including models with a layer of cerebrospinal fluid between the fibers and the brain tissue, are also made. Simulated I₇₈₀ was 0.16 for gray matter, 0.67 for coagulate gray matter, 0.36 for thalamus, 0.39 for coagulated thalamus, unity for white matter, 0.70 for coagulated white matter, and 0.24 for blood. Thalamic matter is also found to reflect more light than gray matter and less than white matter in clinical studies. In conclusion, the reflected light intensity can be used to differentiate between gray and white matter during navigation. Furthermore, coagulation of light gray tissue, such as the thalamus, might be difficult to detect using I₇₈₀, but coagulation in darker gray tissue should result in a rapid increase of I₇₈₀.

We present an automatic method for assessment of pectus excavatum severity based on an optical 3-D markerless shape measurement. A four-directional measurement system based on a structured light projection method is built to capture the shape of the body surface of the patients. The system setup is described and typical measurement parameters are given. The automated data analysis path is explained. Their main steps are: normalization of trunk model orientation, cutting the model into slices, analysis of each slice shape, selecting the proper slice for the assessment of pectus excavatum of the patient, and calculating its shape parameter. We develop a new shape parameter (I_3ds) that shows high correlation with the computed tomography (CT) Haller index widely used for assessment of pectus excavatum. Clinical results and the evaluation of developed indexes are presented.

This PDF file contains the errata for “JBO Vol. 14 Issue 04 Paper JBO ERR-JUL2009-1” for JBO Vol. 14 Issue 04

This PDF file contains the errata for “JBO Vol. 14 Issue 04 Paper JBO ERR-JUL2009-2” for JBO Vol. 14 Issue 04