Conjunctival squamous cell carcinoma (SCC) is an uncommon disease. However, SCC has recently become an important clinical problem due the identification of a significantly high incidence of SCC among a group of black African patients with AIDS. However, basic research concerning SCC, including both intraepithelial and invasive squamous neoplasia, is limited due to the lack of an ocular tumor animal model. Specifically, current ocular imaging and treatment modalities are insufficient for investigating currently available small animal models, because the conjunctival space is not comparable to that of humans. We describe the development of a reproducible model of subconjunctival squamous carcinoma in moderate-sized immunocompetent rabbits. Under optical coherence tomography guidance, 1×10 7 VX2 carcinoma cells are inoculated into the subconjunctival space of 3 to 4-kg New Zealand white rabbits. Malignant tumor involvement developed on the subconjunctival space after an average of 1 to 2 weeks. This subconjunctival tumor model induction method will likely facilitate a broad range of investigation of subconjunctival cancer diagnostics and therapeutics.

We introduce a needle probe based on photoacoustics (PA) to extend the scope of optical needle methods in guiding biopsies. Pulsed light is coupled to an optical fiber in a needle to be inserted in tissue, and PA signals are detected using an ultrasound imager used for needle guidance. This PA needle samples large volumes and possesses an imaging component so that sites forward and off-axis of the fiber are surveyed. This allows navigation of those regions for optical characterization and direct biopsy in a subsequent step. The concept is explored on simple phantoms and biological specimens.

Photoacoustic tomography (PAT) detects ultrasound signals generated by the objects after absorbing illuminating photons. However, the widely used piezoelectric ultrasound transducers are generally not optically transparent, which would cause conflicts between the light illumination and the ultrasonic detection in PAT. We report a different acoustically penetrable optical reflector (APOR) concept to provide a solution to this conflict. We measured the properties of an APOR and experimentally tested its performance in a PAT system. The results demonstrated that the APOR successfully allowed the transducer to detector photoacoustic signals without affecting the light illumination. Moreover, the APOR concept can be readily implemented in various PAT systems.

When using ultrafast laser ablation in some orthopedic applications where precise cutting/drilling is required with minimal damage to collateral tissue, it is challenging to produce large-sized and deep holes using a tightly focused laser beam. The feasibility of producing deep, millimeter-size structures under different ablation strategies is investigated. X-ray computed microtomography was employed to analyze the morphology of these structures. Our results demonstrated the feasibility of producing holes with sizes required in clinical applications using concentric and helical ablation protocols.

Near-infrared (NIR) lasers find applications in neuro-medicine both for diagnostic and treatment purposes. Penetration depth and profile into neural tissue are critical parameters to be considered in these applications. Published data on the optical properties of rodent neural tissue are rare, despite the frequent use of rats as an animal model. The aim of this study was to measure the light intensity profile inside the rat brain using a direct method, while the medium is being illuminated by an NIR laser beam, and compare the results with in vitro measurements of transmittance in the rat brain slices. The intensity profile along the vertical axis had an exponential decline with multiple regions that could be approximated with different coefficients. The Monte Carlo method that was used to simulate light–tissue interactions and predict the scattering coefficient of brain tissue from the measurements suggested that more scattering occurred in deeper layers of the cortex. A single scattering coefficient of 125  cm ⁻¹ was estimated for cortical layers from 300 to 1500 μm and a gradually increasing value from 125 to 370  cm ⁻¹ for depths of 1500 to 3000 μm. The deviations of in vivo results from the in vitro transmittance measurements, as well as the postmortem in vivo results from the alive measurements were significant.

The aim of this work was to evaluate the temperature fields and the dynamics of heat conduction into the skin tissue under several laser irradiation conditions with both a pulsed ultraviolet (UV) laser (λ=337  nm ) and a continuous-wave (cw) visible laser beam (λ=632.8  nm ) using Monte Carlo modeling. Finite-element methodology was used for heat transfer simulation. The analysis of the results showed that heat is not localized on the surface, but is collected inside the tissue in lower skin layers. The simulation was made with the pulsed UV laser beam (used as excitation source in laser-induced fluorescence) and the cw visible laser (used in photodynamic therapy treatments), in order to study the possible thermal effects.

Recent developments in multimodal imaging and image-guided therapy requires multilayered microparticles that encapsulate several imaging and therapeutic agents in the same carrier. However, commonly used microencapsulation processes have multiple limitations such as low encapsulation efficiency and loss of bioactivity for the encapsulated biological cargos. To overcome these limitations, we have carried out both experimental and theoretical studies on coaxial electrospray of multilayered microparticles. On the experimental side, an improved coaxial electrospray setup has been developed. A customized coaxial needle assembly combined with two ring electrodes has been used to enhance the stability of the cone and widen the process parameter range of the stable cone-jet mode. With this assembly, we have obtained poly(lactide-co-glycolide) microparticles with fine morphology and uniform size distribution. On the theoretical side, an instability analysis of the coaxial electrified jet has been performed based on the experimental parameters. The effects of process parameters on the formation of different unstable modes have been studied. The reported experimental and theoretical research represents a significant step toward quantitative control and optimization of the coaxial electrospray process for microencapsulation of multiple drugs and imaging agents in multimodal imaging and image-guided therapy.

A developed temporal focusing-based multiphoton excitation system with additional patterned excitation and local laser control can provide high-throughput fabrication of three-dimensional gray-level biomicrostructures via two-photon cross-linking with rose bengal (RB) as the photoactivator. Multiple bovine serum albumin (BSA) structures of different concentrations were simultaneously achieved by selecting different pulse numbers in the designated regions with an appropriate femtosecond laser power within a few seconds. Intensity of the RB two-photon excited fluorescence (TPEF) is correlated to the concentration of the fabricated cross-linked BSA microstructure. Hence, the fabricated BSA microstructure can be monitored online by utilizing the RB TPEF as contrast agent. It suggests that this approach with its unique capability of high-speed, gray-level, and online-inspection fabrication meets the requirements of the biomedical researches involved in extracellular matrix.

This is the first part of a two-part paper on the application of computer-aided diagnosis to diffuse optical tomography (DOT). An approach for extracting heuristic features from DOT images and a method for using these features to diagnose rheumatoid arthritis (RA) are presented. Feature extraction is the focus of Part 1, while the utility of five classification algorithms is evaluated in Part 2. The framework is validated on a set of 219 DOT images of proximal interphalangeal (PIP) joints. Overall, 594 features are extracted from the absorption and scattering images of each joint. Three major findings are deduced. First, DOT images of subjects with RA are statistically different (p<0.05 ) from images of subjects without RA for over 90% of the features investigated. Second, DOT images of subjects with RA that do not have detectable effusion, erosion, or synovitis (as determined by MRI and ultrasound) are statistically indistinguishable from DOT images of subjects with RA that do exhibit effusion, erosion, or synovitis. Thus, this subset of subjects may be diagnosed with RA from DOT images while they would go undetected by reviews of MRI or ultrasound images. Third, scattering coefficient images yield better one-dimensional classifiers. A total of three features yield a Youden index greater than 0.8. These findings suggest that DOT may be capable of distinguishing between PIP joints that are healthy and those affected by RA with or without effusion, erosion, or synovitis.

This is the second part of a two-part paper on the application of computer-aided diagnosis to diffuse optical tomography (DOT) for diagnosing rheumatoid arthritis (RA). A comprehensive analysis of techniques for the classification of DOT images of proximal interphalangeal joints of subjects with and without RA is presented. A method for extracting heuristic features from DOT images was presented in Part 1. The ability of five classification algorithms to accurately label each DOT image as belonging to a subject with or without RA is analyzed here. The algorithms of interest are the k -nearest-neighbors, linear and quadratic discriminant analysis, self-organizing maps, and support vector machines (SVM). With a polynomial SVM classifier, we achieve 100.0% sensitivity and 97.8% specificity. Lower bounds for these results (at 95.0% confidence level) are 96.4% and 93.8%, respectively. Image features most predictive of RA are from the spatial variation of optical properties and the absolute range in feature values. The optimal classifiers are low-dimensional combinations (<7 features). These results underscore the high potential for DOT to become a clinically useful diagnostic tool and warrant larger prospective clinical trials to conclusively demonstrate the ultimate clinical utility of this approach.

Antibiotic levels in livestock are usually evaluated through destructive analysis. Taking advantage of the fluorescent properties of marbofloxacin (MBX) and trovafloxacin (TVX), multiphoton microscopy (MPM) was evaluated as a minimally invasive and nondestructive method to determine the penetration of TVX and MBX into sheep neutrophils. Standard curves were measured with drug-only solutions and suggested that MBX was more suited for this type of analysis. The intracellular concentration of both TVX and MBX was higher than the extracellular concentration after incubating neutrophils for 30 min at concentrations ranging from 0.1 to 100  μg/ml for both the drugs. The intracellular concentration of TVX increased with the extracellular concentration but was always greater than the extracellular concentration, suggesting active internalization. On the other hand, intracellular/extracellular ratio (I/E) peaked at 1.6-fold I/E for 1  μg/ml and then gradually decreased with increased concentration to 1.2-fold I/E at 100  μg/ml . For the first time, this study showed the use of MPM to quantify antibiotic uptake by sheep neutrophils and observed that both antibiotics were taken up by sheep neutrophils beyond extracellular levels.

Innovative luminescent nanomaterials, termed upconversion nanoparticles (UCNPs), have demonstrated considerable promise as molecular probes for high-contrast optical imaging in cells and small animals. The feasibility study of optical diagnostics in humans is reported here based on experimental and theoretical modeling of optical imaging of an UCNP-labeled breast cancer lesion. UCNPs synthesized in-house were surface-capped with an amphiphilic polymer to achieve good colloidal stability in aqueous buffer solutions. The scFv4D5 mini-antibodies were grafted onto the UCNPs via a high-affinity molecular linker barstar:barnase (Bs:Bn) to allow their specific binding to the human epidermal growth factor receptor HER2/neu, which is overexpressed in human breast adenocarcinoma cells SK-BR-3. UCNP-Bs:Bn-scFv4D5 biocomplexes exhibited high-specific immobilization on the SK-BR-3 cells with the optical contrast as high as 10:1 benchmarked against a negative control cell line. Breast cancer optical diagnostics was experimentally modeled by means of epi-luminescence imaging of a monolayer of the UCNP-labeled SK-BR-3 cells buried under a breast tissue mimicking optical phantom. The experimental results were analyzed theoretically and projected to in vivo detection of early-stage breast cancer. The model predicts that the UCNP-assisted cancer detection is feasible up to 4 mm in tissue depth, showing considerable potential for diagnostic and image-guided surgery applications.

The in vitro and in vivo detection of visible photons from radioisotopes using optical techniques is a fast-growing field in molecular imaging. Tc 99m -pertechnetate is used as an alternative to I 123 in imaging of the thyroid and is generally imaged with gamma cameras or single photon emission tomography instruments. The uptake in the thyroid tissue is mediated by the sodium-iodide symporter (NIS), a glycoprotein that actively mediates iodide transport into the thyroid follicular cells and several extrathyroidal tissues. The luminescence of the gamma emitter Tc 99m -pertechnetate in order to visualize its biodistribution in healthy small living animals by using a commercial optical imaging system is investigated. Here we show that in Nu/Nu mice, the uptake of Tc 99m -pertechnetate in the thyroid gland and in salivary glands is very detectable by using radionuclide luminescence imaging. We also found light emission from the stomach in accordance with the literature. The localization of the light signals in the anatomical regions where the radiopharmaceutical is expected, confirmed by resections, shows that it is possible to image NIS-expressing tissues.

Spectral-domain optical coherence tomography (SD-OCT) is a three-dimensional imaging technique that allows direct visualization of retinal morphology and architecture. The various retinal layers may be affected differentially by various diseases. An automated graph search algorithm is developed to sequentially segment 11 retinal surfaces in SD-OCT volumes using a three-stage approach. In stage 1, the four most easily discernible and/or distinct surfaces are identified in four-times-downsampled images and are used as a priori information to limit the graph search for the other surfaces in stage 2. Eleven surfaces were then detected in two-times-downsampled images in stage 2, and refined in the original images in stage 3 using the graph search integrating the estimated morphological shape models. Twenty macular SD-OCT volume scans from 20 normal subjects are used in this initial study. The overall mean and absolute mean differences in border positions between the automated and manual segmentation for the 11 surfaces are −0.20±0.53 voxels (−0.76±2.06  μm ) and 0.82±0.64 voxels (3.19±2.46  μm ), respectively. Intensity/reflectivity and thickness properties in various retinal layers are also investigated. This investigation in normal subjects may provide a comparative reference for subsequent adaptations in eyes with diseases.

Vasoactive drugs are normally utilized to elevate mean artery pressure and maintain adequate organ perfusion in clinical treatment. During the injection, morphological changes and the subsequent oxygen supply alteration in the brain, e.g., possible hypoxia, are prone to introduce serious damage and even dysfunction to the brain. Therefore, multiparameter monitoring of cerebral microvasculature is necessary during drug injection. An optical-resolution photoacoustic microscopy was used to assess the effects of norepinephrine on microvasculature in the brain cortex of mice. In our experiments, the diameter, total hemoglobin (HbT) and oxygen saturation (SO ₂ ) of single cerebral microvessels during tail vein injection of norepinephrine were analyzed. Following the injection, vasoconstriction was observed, and HbT and SO ₂ were decreased in turn. The vessel diameter and HbT recovered back to the base value without further injection, while the SO ₂ remained low throughout the observation period. Arterioles showed more acute constriction but a smaller decline in HbT during the injection compared with venules, while SO ₂ in arterioles increased slightly without further drug injection but not in venules. Our results suggested that photoacoustic microscopy may become a new method for early and comprehensive evaluation of the effect of drugs on microvasculature in brain.

An optical coherence tomography (OCT) microvascular imaging platform, consisting of Doppler (DOCT) and speckle variance (svOCT) modalities, and microvascular image quantification tools are developed. The quantification methods extract blood flow-related parameters from DOCT images and vessel morphological parameters from svOCT images. This platform is used to assess the microvascular (DOCT and svOCT) images obtained during a clinical study on late oral radiation toxicity. This specific pathology was considered a suitable scenario for verifying the performance of the developed quantification platform because late oral radiation toxicity is known to involve microvascular damage. The derived parameters are compared between several DOCT and svOCT images from one patient and one healthy volunteer as proof-of-principle, and the significance of the observed differences is discussed. Given the low number of OCT clinical studies that measure and quantify microvascular images and considering the importance of such quantification in a number of pathologies, this newly developed platform can serve as a useful tool in studying diseases and treatments with microvascular involvement.

A multimodal multiphoton nonlinear optical (NLO) microspectroscopy imaging system was developed using a femtosecond laser and a photonic crystal fiber. Coherent anti-Stokes Raman scattering (CARS) microspectroscopy was combined with two-photon excitation fluorescence and second-harmonic generation microscopy in one platform and the system was applied to diagnose liver fibrosis. Normal and liver fibrosis tissues were clearly distinguished with the great difference from CARS spectra as well as multimodal multiphoton NLO images. We expect the system to be a rapid diagnosis tool for liver fibrosis at tissue level with label-free imaging of significant biochemical components.

A novel approach is presented for obtaining fast robust three-dimensional (3-D) reconstructions of bioluminescent reporters buried deep inside animal subjects from multispectral images of surface bioluminescent photon densities. The proposed method iteratively acts upon the equations relating the multispectral data to the luminescent distribution with high computational efficiency to provide robust 3-D reconstructions. Unlike existing algebraic reconstruction techniques, the proposed method is designed to use adaptive projections that iteratively guide the updates to the solution with improved speed and robustness. Contrary to least-squares reconstruction methods, the proposed technique does not require parameter selection or optimization for optimal performance. Additionally, optimized schemes for thresholding, sampling, and ordering of the bioluminescence tomographic data used by the proposed method are presented. The performance of the proposed approach in reconstructing the shape, volume, flux, and depth of luminescent inclusions is evaluated in a multitude of phantom-based and dual-modality in vivo studies in which calibrated sources are implanted in animal subjects and imaged in a dual-modality optical/computed tomography platform. Statistical analysis of the errors in the depth and flux of the reconstructed inclusions and the convergence time of the proposed method is used to demonstrate its unbiased performance, low error variance, and computational efficiency.

The dynamic vascular changes in the breast resulting from manipulation of both inspired end-tidal partial pressure of oxygen and carbon dioxide were imaged using a 30 s per frame frequency-domain near-infrared spectral (NIRS) tomography system. By analyzing the images from five subjects with asymptomatic mammography under different inspired gas stimulation sequences, the mixture that maximized tissue vascular and oxygenation changes was established. These results indicate maximum changes in deoxy-hemoglobin, oxygen saturation, and total hemoglobin of 21, 9, and 3%, respectively. Using this inspired gas manipulation sequence, an individual case study of a subject with locally advanced breast cancer undergoing neoadjuvant chemotherapy (NAC) was analyzed. Dynamic NIRS imaging was performed at different time points during treatment. The maximum tumor dynamic changes in deoxy-hemoglobin increased from less than 7% at cycle 1, day 5 (C1, D5) to 17% at (C1, D28), which indicated a complete response to NAC early during treatment and was subsequently confirmed pathologically at the time of surgery.

Fluorescence recovery after photobleaching (FRAP) is a common technique to probe mobility of fluorescently labeled proteins in biological membranes by monitoring the time-dependence of the spatially integrated fluorescence signals after a bleaching pulse. Discrimination by FRAP between free diffusion with an immobile fraction (FDIM) and the phenomenological model for anomalous diffusion based on the time-dependent diffusion coefficient (TDDC) is a challenging problem, requiring extremely long observation times for differentiation. Recently, rectangular FRAP (rFRAP) has been introduced for normal diffusion by considering not only the temporal but also spatial information, taking the effective point spread function of the optical system into account. In this work we provide an extension of rFRAP toward anomalous diffusion according to the continuous time random walk (CTRW). We explore whether the spatial information in rFRAP allows for enhanced discrimination between FDIM, TDDC, and CTRW in a single experiment within a feasible time window. Simulations indicate that rFRAP can indeed differentiate the different models by evaluating the spatial autocorrelation of the differences between the measured and fitted pixel values. Hence, rFRAP offers a tool that is capable of discriminating different types of diffusion at shorter time scales than in the case where spatial information is discarded.

Quantitative spectroscopy has recently been extended from a contact-probe to wide-area spectroscopic imaging to enable mapping of optical properties across a wide area of tissue. We train quantitative spectroscopic imaging (QSI) to identify cervical high-grade squamous intraepithelial lesions (HSILs) in 34 subjects undergoing the loop electrosurgical excision procedure (LEEP subjects). QSI’s performance is then prospectively evaluated on the clinically suspicious biopsy sites from 47 subjects undergoing colposcopic-directed biopsy. The results show the per-subject normalized reduced scattering coefficient at 700 nm (A _n) and the total hemoglobin concentration are significantly different (p<0.05 ) between HSIL and non-HSIL sites in LEEP subjects. A _n alone retrospectively distinguishes HSIL from non-HSIL with 89% sensitivity and 83% specificity. It alone applied prospectively on the biopsy sites distinguishes HSIL from non-HSIL with 81% sensitivity and 78% specificity. The findings of this study agree with those of an earlier contact-probe study, validating the robustness of QSI, and specifically A _n , for identifying HSIL. The performance of A _n suggests an easy to use and an inexpensive to manufacture monochromatic instrument is capable of early cervical cancer detection, which could be used as a screening and diagnostic tool for detecting cervical cancer in low resource countries.

Cross sectional tomographic systems based on cylindrically focused transducers are widely used in optoacoustic (photoacoustic) imaging due to important advantages they provide such as high-cross sectional resolution, real-time imaging capacity, and high-throughput performance. Tomographic images in such systems are commonly obtained by means of two-dimensional (2-D) reconstruction procedures assuming point-like detectors, and volumetric (whole-body) imaging is performed by superimposing the cross sectional images for different positions along the scanning direction. Such reconstruction strategy generally leads to in-plane and out-of-plane artifacts as well as significant quantification errors. Herein, we introduce two equivalent full three-dimensional (3-D) models capable of accounting for the shape of cylindrically focused transducers. The performance of these models in 3-D reconstructions considering several scanning positions is analyzed in this work. Improvements of the results rendered with the introduced reconstruction procedure as compared with the 2-D-based approach are described and discussed for simulations and experiments with phantoms and biological tissues.

Confocal scanning laser ophthalmoscopy (cSLO) enables high-resolution and high-contrast imaging of the retina by employing spatial filtering for scattered light rejection. However, to obtain optimized image quality, one must design the cSLO around scanner technology limitations and minimize the effects of ocular aberrations and imaging artifacts. We describe a cSLO design methodology resulting in a simple, relatively inexpensive, and compact lens-based cSLO design optimized to balance resolution and throughput for a 20-deg field of view (FOV) with minimal imaging artifacts. We tested the imaging capabilities of our cSLO design with an experimental setup from which we obtained fast and high signal-to-noise ratio (SNR) retinal images. At lower FOVs, we were able to visualize parafoveal cone photoreceptors and nerve fiber bundles even without the use of adaptive optics. Through an experiment comparing our optimized cSLO design to a commercial cSLO system, we show that our design demonstrates a significant improvement in both image quality and resolution.

Fluorescence diffuse optical tomography (fDOT) is a noninvasive imaging technique that makes it possible to quantify the spatial distribution of fluorescent tracers in small animals. fDOT image reconstruction is commonly performed by means of iterative methods such as the algebraic reconstruction technique (ART). The useful results yielded by more advanced l ₁ -regularized techniques for signal recovery and image reconstruction, together with the recent publication of Split Bregman (SB) procedure, led us to propose a new approach to the fDOT inverse problem, namely, ART-SB. This method alternates a cost-efficient reconstruction step (ART iteration) with a denoising filtering step based on minimization of total variation of the image using the SB method, which can be solved efficiently and quickly. We applied this method to simulated and experimental fDOT data and found that ART-SB provides substantial benefits over conventional ART.

While color video endoscopy has enabled wide-field examination of the gastrointestinal tract, it often misses or incorrectly classifies lesions. Many of these missed lesions exhibit characteristic three-dimensional surface topographies. An endoscopic system that adds topographical measurements to conventional color imagery could therefore increase lesion detection and improve classification accuracy. We introduce photometric stereo endoscopy (PSE), a technique which allows high spatial frequency components of surface topography to be acquired simultaneously with conventional two-dimensional color imagery. We implement this technique in an endoscopic form factor and demonstrate that it can acquire the topography of small features with complex geometries and heterogeneous optical properties. PSE imaging of ex vivo human gastrointestinal tissue shows that surface topography measurements enable differentiation of abnormal shapes from surrounding normal tissue. Together, these results confirm that the topographical measurements can be obtained with relatively simple hardware in an endoscopic form factor, and suggest the potential of PSE to improve lesion detection and classification in gastrointestinal imaging.

In clinical dental practice, it is often difficult or even impossible to detect and assess interfacial adhesive defects at adhesive restorations by means of visual inspection or other established diagnostic methods. However, nondestructive optical coherence tomography (OCT) may provide a better picture in this diagnostic scenario. The aim of this study was to evaluate the suitability of swept source OCT (SS-OCT) for the nondestructive assessment of interfacial deficiencies at composite restorations and the evaluation of cohesive defects within composite material. Ten class V composite restorations that were not adhesively luted were taken as validation objects and examined for frequency of interfacial gaps, air entrapments, and defects between composite layers using SS-OCT with a 1325-nm center wavelength. Light microscopy was used to inspect for inherent structures. SS-OCT detected 79.5%±1.8% of the total gap lengths at the enamel interface and 70.9%±0.4% at the dentin interface. Additionally, defective structures in composite restorations were displayed. It was shown that OCT imaging has the potential to nondestructively assess the interfacial adaptation of composite restorations and to detect internal defects in the layered composite material.

Tumor hypoxia is an important factor in assessment of both cancer progression and cancer treatment efficacy. This has driven a substantial effort toward development of imaging modalities that can directly measure oxygen distribution and therefore hypoxia in tissue. Although several approaches to measure hypoxia exist, direct measurement of tissue oxygen through an imaging approach is still an unmet need. To address this, we present a new approach based on in vivo application of photoacoustic lifetime imaging (PALI) to map the distribution of oxygen partial pressure (pO² ) in tissue. This method utilizes methylene blue, a dye widely used in clinical applications, as an oxygen-sensitive imaging agent. PALI measurement of oxygen relies upon pO² -dependent excitation lifetime of the dye. A multimodal imaging system was designed and built to achieve ultrasound (US), photoacoustic, and PALI imaging within the same system. Nude mice bearing LNCaP xenograft hindlimb tumors were used as the target tissue. Hypoxic regions were identified within the tumor in a combined US/PALI image. Finally, the statistical distributions of pO² in tumor, normal, and control tissues were compared with measurements by a needle-mounted oxygen probe. A statistically significant drop in mean pO² was consistently detected by both methods in tumors.

This study aimed to nondestructively evaluate sealing performance of eight one-step self-etch adhesives (1-SEAs) using optical coherence tomography (OCT). The two-step self-etch adhesive (2-SEA) served as the control. Round tapered class-I cavities (D=4  mm , H=2  mm ) were prepared in bovine incisors, treated with each adhesive (n=5 ), and restored with a flowable resin composite. Cross-sections were obtained from each restoration using swept-source OCT with 1310-nm laser. The average percentage of the sealed interface (SI%) for each adhesive was calculated using image analysis software, considering increased signal intensity at the interface as gap. Samples were then sectioned and observed under confocal laser scanning microscope (CLSM). Significantly different SI% values were found among different adhesives (analysis of variance, Bonferroni, p<0.05 ). There was also a significant correlation in SI% between OCT and CLSM (p<0.0001 , r=0.96 ). Additionally, microscopic analysis revealed that the gaps in 1-SEAs occurred not only at dentine–adhesive interfaces but also frequently at adhesive–composite interfaces. Some recent 1-SEAs could achieve reliable short-term sealing comparable to 2-SEA. OCT is a unique tool to nondestructively evaluate the sealing performance of the restoratives through the cavity, provided that cavity walls have a certain minimum inclination with respect to the beam.

The potential of intrinsic optical imaging and resting-state analysis under anesthetized conditions as a tool to study brain networks associated with epileptic seizures is investigated. Using an acute model of epileptiform activity, the 4-aminopyridine model in live mice, we observe the changes in resting-state networks with the onset of seizure activity and in conditions of spiking activity. Resting-state networks identified before and after the onset of epileptiform activity show both decreased and increased homologous correlations, with a small dependence on seizure intensity. The observed changes are not uniform across the different hemodynamic measures, suggesting a potential decoupling between blood flow and metabolism in the low-frequency networks. This study supports the need for a more extensive investigation of epileptic networks including more than one independent hemodynamic measurement.

The seizure onset zone (SOZ) and propagation pathways in supplementary motor area (SMA) seizures are difficult to identify because of the short duration and swift propagations of this seizure type. Herein, we studied ictal cerebral blood flow changes, seizure electrical activities, and clinical signs employing simultaneous near-infrared spectroscopy (NIRS) and electrocorticography (ECoG) recordings in a brain tumor patient with SMA seizures. Increased cerebral blood flow was observed from the SOZ in the ipsilateral SMA and spread to the ipsilateral premotor cortex, ipsilateral sensorimotor cortex, and also the contralateral hemisphere. These propagation patterns were concordant with ictal ECoG seizure activities and clinical signs. Removal of the SOZ and surrounding areas, where the higher blood flow and higher frequency oscillations had been identified, achieved a good outcome for this patient. Our ictal NIRS-ECoG technique is robust and useful for detecting the SOZ and seizure propagations.

Actinic cheilitis (AC) is a potentially malignant disorder of the lips. Because of its heterogeneous clinical aspect, it is difficult to indicate representative biopsy area. The purpose of this study was to evaluate the usefulness of tissue autofluorescence in AC diagnosis. The system was composed of a 405-nm light-emitting diode, sent to the sample by a dichroic, that allows the fluorescence signal to reach a camera directly plugged in the system. Fifty-seven patients with clinical diagnosis of AC and 45 normal volunteers were selected. According to clinical and fluorescence features, one or more areas were selected for biopsies in the AC group and epithelial dysplasia (ED) grades were established. The autofluorescence images were processed by a clustering algorithm for AC automated diagnosis. The tissue autofluorescence image revealed a heterogeneous pattern of loss and increase of fluorescence in patients with AC. ED was found in 93% of the cases, and most of the areas graded as moderate or severe ED were chosen with the aid of autofluorescence. The processed autofluorescence images from AC patients showed a higher number of spots in an irregular pattern. Tissue autofluorescence image system is a useful technique in association with clinical examination for AC diagnosis.

Stroke is a worldwide medical emergency and an important issue in stroke research is looking for the early pathophysiological markers which can predict the severity of brain injury. Decreased cerebral blood flow (CBF) has been serving as the most important indicator of ischemic stroke. Particular attention is paid to study the spatio-temporal CBF changes immediately after the onset of stroke in a rat intraluminal filament middle cerebral artery occlusion (MCAO) model and investigation of its correlation with brain infarct volume after 24 h. We implement an on-line laser speckle imaging (LSI) system, which could provide real time high spatio-temporal resolution CBF information before, during, and immediately after the rat MCAO surgery. We found a significant correlation between the affected area with 50% CBF reduction (CBF₅₀ ) at the first minute after occlusion with the infarct volume. To the best of our knowledge, this is the earliest CBF marker for infarct volume prediction. Based on such a CBF–infarct volume correlation, LSI may be used as a real time guidance for improving the consistency of intraluminal filament MCAO model since the depth of filament insertion could be adjusted promptly and those unsuccessful models could be excluded in the earliest stage.

High-resolution angiography and total average blood flow measurements in the rat retina using optical coherence tomography (OCT) are presented. Recently, an en face integration method, which does not require explicit calculation of vessel angles, was introduced to calculate blood flow using Doppler OCT. Using this method, rapid two-dimensional scanning has been shown to quantify pulsatile flow. However, high scanning speeds sacrifice transverse sampling density and may lead to biased velocity estimates. Alternatively, we show here that by using a volumetric scanning protocol that asynchronously samples a single vessel with respect to the heartbeat, it is possible to obtain accurate time-averaged flow measurements, even without explicit information about the pulsatile waveform. Total average retinal blood flows calculated using either arteries or veins are comparable, supporting the accuracy of this method.

Clinical diagnoses of bone health and fracture risk typically rely on measurements of bone density or structure, but the strength of a bone is also dependent on its chemical composition. Raman spectroscopy has been used extensively in ex vivo studies to measure the chemical composition of bone. Recently, spatially offset Raman spectroscopy (SORS) has been utilized to measure bone transcutaneously. Although the results are promising, further advancements are necessary to make noninvasive, in vivo measurements of bone with SORS that are of sufficient quality to generate accurate predictions of fracture risk. In order to separate the signals from bone and soft tissue that contribute to a transcutaneous measurement, we developed an overconstrained extraction algorithm that is based on fitting with spectral libraries. This approach allows for accurate spectral unmixing despite the fact that similar chemical components (e.g., type I collagen) are present in both bone and soft tissue. The algorithm was utilized to transcutaneously detect biochemical differences in the tibiae of wild-type mice between 1 and 7 months of age and between the tibiae of wild-type mice and a mouse model of osteogenesis imperfecta. These results represent the first diagnostically sensitive, transcutaneous measurements of bone using SORS.

Detection and enumeration of rare circulating cells in mice are important problems in many areas of preclinical biomedical research. Recently, we developed a new method termed “diffuse fluorescence flow cytometry” (DFFC) that uses diffuse photons to increase the blood sampling volume and sensitivity versus existing in vivo flow cytometry methods. In this work, we describe a new DFFC prototype with approximately an order-of-magnitude improvement in sensitivity compared to our previous work. This sensitivity improvement is enabled by a number of technical innovations, which include a method for the removal of motion artifacts (allowing interrogation of mouse hindlegs that was less optically attenuating versus the tail) and improved collection optics and signal preamplification. We validated our system first in limb mimicking optical flow phantoms with fluorescent microspheres and then in nude mice with fluorescently labeled mesenchymal stem cells at injected concentrations of 5×10 ³   cells/mL . In combination, these improvements resulted in an overall cell counting sensitivity of about 1  cell/mL or better in vivo.

The high incidence and mortality of breast cancer requires an effective, rapid, and cost-effective method for its diagnosis. Here, visible and near-infrared spectroscopy in the wavelength range of 400 to 2200 nm is utilized for distinguishing the malignant tumor tissue from benign tumor and normal breast tissues. Based on the absorption and scattering spectra of fixed samples, three spectral analysis methods are proposed which include an absorption spectral analysis, a scattering spectral analysis, and a combined spectral analysis of the two. By comparison with the histopathological examination, the sensitivity, specificity, and accuracy of the three analysis methods are calculated. The results showed that the combined spectral analysis method can significantly enhance the effectiveness when compared with the sole absorption or scattering spectral analysis method. The sensitivity, specificity, and accuracy of the combined spectral analysis method are 100%, 87.82%, and 87.50% for the benign tumor tissue and 81.82%, 100%, and 87.5% for malignant tumor tissue, respectively. All of the th8ree values are 100% for normal breast tissue. This study demonstrates that the combined spectral analysis method has better potential for in vitro optical diagnosis for breast lesions.

A diagnosis criterion is proposed for noninvasive grading of burn injuries using terahertz radiation. Experimental results are presented from in vivo terahertz time-domain spectroscopy of second- and third-degree wounds, which are obtained in a 72-hour animal study. During this period, the change in the spectroscopic response of the burned tissue is studied. It is shown that terahertz waves are sensitive not only to the postburn formation of interstitial edema, but also to the density of skin structures derived from image processing analysis of histological sections. Based on these preliminary results, it is suggested that the combination of these two effects, as probed by terahertz spectroscopy of the tissue, may ultimately be used to differentiate partial-thickness burns that will naturally heal from those that will require surgical intervention.

Gold nanoparticles can be used as molecular contrast agents binding specifically to cancer sites and thus delineating tumor regions. Imaging gold nanoparticles deeply embedded in tissues with optical techniques possesses significant challenges due to multiple scattering of optical photons that blur the obtained images. Both diagnostic and therapeutic applications can benefit from a minimally invasive technique that can identify, localize, and quantify the payloads of gold nanoparticles deeply embedded in biological tissues. An optical radiance technique is applied to map localized inclusions of gold nanorods in 650- to 900-nm spectral range in a porcine phantom that mimics prostate geometry. Optical radiance defines a variation in the angular density of photons impinging on a selected point in the tissue from various directions. The inclusions are formed by immersing a capillary filled with gold nanorods in the phantom at increasing distances from the detecting fiber. The technique allows the isolation of the spectroscopic signatures of the inclusions from the background and identification of inclusion locations in the angular domain. Detection of ∼4×10 ¹⁰ gold nanoparticles or 0.04  mg Au/mL (detector–inclusion separation 10 mm, source–detector separation 15 mm) in the porcine tissue is demonstrated. The encouraging results indicate a promising potential of radiance spectroscopy in early prostate cancer diagnostics with gold nanoparticles.

The use of thulium fiber laser (TFL) as a potential alternative laser lithotripter to the clinical holmium:YAG laser is being studied. The TFL’s Gaussian spatial beam profile provides efficient coupling of higher laser power into smaller core fibers without proximal fiber tip degradation. Smaller fiber diameters are more desirable, because they free up space in the single working channel of the ureteroscope for increased saline irrigation rates and allow maximum ureteroscope deflection. However, distal fiber tip degradation and “burn-back” increase as fiber diameter decreases due to both excessive temperatures and mechanical stress experienced during stone ablation. To eliminate fiber tip burn-back, the distal tip of a 150-μm core silica fiber was glued inside 1-cm-long steel tubing with fiber tip recessed 100, 250, 500, 1000, or 2000 μm inside the steel tubing to create the hollow-tip fiber. TFL pulse energy of 34 mJ with 500-μs pulse duration and 150-Hz pulse rate was delivered through the hollow-tip fibers in contact with human calcium oxalate monohydrate urinary stones during ex vivo studies. Significant fiber tip burn-back and degradation was observed for bare 150-μm core-diameter fibers. However, hollow steel tip fibers experienced minimal fiber burn-back without compromising stone ablation rates. A simple, robust, compact, and inexpensive hollow fiber tip design was characterized for minimizing distal fiber burn-back during the TFL lithotripsy. Although an increase in stone retropulsion was observed, potential integration of the hollow fiber tip into a stone basket may provide rapid stone vaporization, while minimizing retropulsion.

The excitation-related problems in photodynamic therapy of cancer might be solved by combining two-photon (TP) irradiation and quantum dots (QDs) as effective energy donors for conventional photosensitizers (PS). Here, it is demonstrated for the first time that QD–chlorin e ₆ (Ce ₆ ) complex formed due to the hydrophobic interaction between Ce ₆ molecules and lipid coating of QDs can be effectively excited via TP irradiation at 1030 nm, which spectrally coincides with the biological tissue optical window. TP absorption cross-section for free QDs and Ce ₆ at 1030 nm was 3325 and 13 Goeppert-Mayer, respectively. Upon TP excitation of QD−Ce ₆ solution, the fluorescence band of bound Ce ₆ molecules was observed via energy transfer from excited QDs. Increasing concentration of Ce 6 resulted in quenching of the photoluminescence of QDs and an increase in the fluorescence intensity of bound Ce ₆ molecules. These intensity changes coincided well with those observed upon single-photon excitation of QD−Ce ⁶ solution when QDs alone are excited. The efficiency of energy transfer in QD−Ce ₆ complex upon TP excitation was about 80% (QD∶Ce ⁶ 1∶5 ). These results indicate that the effective excitation of PS with a low TP absorption cross-section is possible in such type noncovalent complexes via energy transfer from TP excited QDs.

This article [J. Biomed. Opt.. 18, , 061226 (2013)] was originally published online on 6 February 2013 with an error in the article’s title. The third word should be “nanorods’” instead of “nanoroad.” The corrected title appears above.

It is fascinating how optical technology transfers among disparate fields of science and engineering. The history of adaptive optics is a good case study. The story begins in 1953 when a visionary, Horace Babcock, who was an astronomer at the Mount Wilson and the Palomar observatories, proposed a method based on adaptive optics to correct in real time the atmospheric distortions that degraded ground-based telescope images. All ground-based telescopes suffer from atmospheric turbulence, which causes time-dependent inhomogeneities in the air refractive index. They are caused by nonstationary random processes. The wind shears mix various atmospheric layers and the temperature inhomogeneities result in time-dependent variations in the refractive index of the air. They distort the wavefronts and thus degrade the image. One alternative is space-based telescopes such as the Hubble Space Telescope. Another is to implement Babcock’s idea of a closed-loop system incorporating a wavefront sensor and a deformable mirror that can introduce real-time changes in the wavefront to compensate for the aberrations introduced by the atmospheric turbulence. Babcock’s prescient ideas were developed into instrumentation in the mid-1970s and in 1982 the Defense Advanced Research Projects Agency (DARPA) working with the United States Air Force completed a real-time adaptive optics system integrated with an optical telescope on Maui in Hawaii. The motivation was to obtain high-resolution images of Soviet satellites. One common wavefront sensor is the Shack-Hartmann wavefront sensor, which works with white light and also with extended sources (such as the Sun). The closed-loop adaptive optics system involves computer wavefront reconstruction, which is a classical inverse problem whose solution can be found, but the solution cannot be proved to be unique.