A high-speed optical coherence tomography (OCT) with 1-μm axial resolution was applied to assess the thickness of a cell-free layer (CFL) and a spatial distribution of red blood cells (RBC) next to the microchannel wall. The experiments were performed in vitro in a plain glass microchannel with a width of 2 mm and height of 0.2 mm. RBCs were suspended in phosphate buffered saline solution at the hematocrit level of 45%. Flow rates of 0.1 to 0.5  ml/h were used to compensate gravity induced CFL. The results indicate that OCT can be efficiently used for the quantification of CFL thickness and spatial distribution of RBCs in microcirculatory blood flow.

Although fetal scalp blood sampling is an examination to assess fetal acidosis during the intrapartum period, it has not been widely used by obstetricians because of its invasiveness. We have developed a small, portable oximetry with a sensor attached to the examiner’s finger. Our previous report using this oximetry concluded that fetal head tissue oxygen saturation (StO₂) correlated with umbilical cord artery blood pH. We investigated whether the association between StO₂ and blood pH in mice could be validated using this oximetry. Eleven the Institute for Cancer Research (ICR) mice were measured using a near-infrared spectroscopy probe at the craniofacial site in a closed polyethylene bag while changing the oxygen concentration. A total of nine blood samples were collected and analyzed for pH. The StO₂ and tissue blood pH showed a strong positive correlation (r=0.90 and P=0.0009). The StO₂ and total hemoglobin index also showed a positive correlation (r=0.84 and P=0.0049). Thus, the results of the present study support those of our previous report on clinical cases and allow examiners to easily check the status of fetal acidosis. Fetal management using this oximetry might gain popularity with obstetricians in the near future.

An algorithm for the simulation of image formation in Fourier domain optical coherence tomography (OCT) for an infinitely long cylinder is presented. The analytical solution of Maxwell’s equations for light scattering by a single cylinder is employed for the case of perpendicular incidence to calculate OCT images. The A-scans and the time-resolved scattered intensities are compared to geometrical optics results calculated with a ray tracing approach. The reflection peaks, including the whispering gallery modes, are identified. Additionally, the Debye series expansion is employed to identify single peaks in the OCT A-scans. Furthermore, a Gaussian beam is implemented in order to simulate lateral scanning over the cylinder for two-dimensional B-scans. The fields are integrated over a certain angular range to simulate a detection aperture. In addition, the solution for light scattering by layered cylinders is employed and the various layers are identified in the resulting OCT image. Overall, the simulations in this work show that OCT images do not always display the real surface of investigated samples.

We performed near-diffraction limited two-photon fluorescence (TPF) imaging through a lensless, multicore-fiber (MCF) endoscope utilizing digital phase conjugation. The phase conjugation technique is compatible with commercially available MCFs with high core density. We demonstrate focusing of ultrashort pulses through an MCF and show that the method allows for resolution that is not limited by the MCF core spacing. We constructed TPF images of fluorescent beads and cells by digital scanning of the phase-conjugated focus on the target object and collection of the emitted fluorescence through the MCF.

We highlight the options available for noninvasive optical diagnostics of reporter gene expression in mouse tibialis cranialis muscle. An in vivo multispectral imaging technique combined with fluorescence spectroscopy point measurements has been used for the transcutaneous detection of enhanced green fluorescent protein (EGFP) expression, providing information on location and duration of EGFP expression and allowing quantification of EGFP expression levels. For EGFP coding plasmid (pEGFP-Nuc Vector, 10  μg/50  ml) transfection, we used electroporation or ultrasound enhanced microbubble cavitation [sonoporation (SP)]. The transcutaneous EGFP fluorescence in live mice was monitored over a period of one year using the described parameters: area of EGFP positive fibers, integral intensity, and mean intensity of EGFP fluorescence. The most efficient transfection of EGFP coding plasmid was achieved, when one high voltage and four low voltage electric pulses were applied. This protocol resulted in the highest short-term and long-term EGFP expression. Other electric pulse protocols as well as SP resulted in lower fluorescence intensities of EGFP in the transfected area. We conclude that noninvasive multispectral imaging technique combined with fluorescence spectroscopy point measurements is a suitable method to estimate the dynamics and efficiency of reporter gene transfection in vivo.

This study aimed to determine the feasibility of using optical coherence elastography to measure internal displacements during the curing phase of a light-activated, resin-based composite material. Displacement vectors were spatially mapped over time within a commercial dental composite. Measurements revealed that the orientation of cure-induced displacement vectors varied spatially in a complex manner; however, each vector showed a systematic evolution with time. Precision of individual displacements was estimated to be ∼1 to 2  μm, enabling submicrometer time-varying displacements to be detected.

Systemic sclerosis (SSc) is a connective tissue disease that results in excessive accumulation of collagen in the skin and internal organs. Overall, SSc has a rare morbidity (276 cases per million adults in the United States), but has a 10-year survival rate of 55%. Currently, the modified Rodnan skin score (mRSS) is assessed by palpation on 17 sites on the body. However, the mRSS assessed score is subjective and may be influenced by the experience of the rheumatologists. In addition, the inherent elasticity of skin may bias the mRSS assessment in the early stage of SSc, such as oedematous. Optical coherence elastography (OCE) is a rapidly emerging technique, which can assess mechanical contrast in tissues with micrometer spatial resolution. In this work, the OCE technique is applied to assess the mechanical properties of skin in both control and bleomycin (BLM) induced SSc-like disease noninvasively. Young’s modulus of the BLM-SSc skin was found be significantly higher than that of normal skin, in both the in vivo and in vitro studies (p<0.05). Thus, OCE is able to differentiate healthy and fibrotic skin using mechanical contrast. It is a promising new technology for quantifying skin involvement in SSc in a rapid, unbiased, and noninvasive manner.

Indocyanine green (ICG) fluorescence imaging has been clinically used for noninvasive visualizations of vascular structures. We have previously developed a diagnostic system based on dynamic ICG fluorescence imaging for sensitive detection of vascular disorders. However, because high-dimensional raw data were used, the analysis of the ICG dynamics proved difficult. We used principal component analysis (PCA) in this study to extract important elements without significant loss of information. We examined ICG spatiotemporal profiles and identified critical features related to vascular disorders. PCA time courses of the first three components showed a distinct pattern in diabetic patients. Among the major components, the second principal component (PC2) represented arterial-like features. The explained variance of PC2 in diabetic patients was significantly lower than in normal controls. To visualize the spatial pattern of PCs, pixels were mapped with red, green, and blue channels. The PC2 score showed an inverse pattern between normal controls and diabetic patients. We propose that PC2 can be used as a representative bioimaging marker for the screening of vascular diseases. It may also be useful in simple extractions of arterial-like features.

To facilitate regular assessment of the microcirculation in vivo, noninvasive imaging techniques such as nailfold capillaroscopy are required in clinics. Recently, a correlation mapping technique has been applied to optical coherence tomography (OCT), which extends the capabilities of OCT to microcirculation morphology imaging. This technique, known as correlation mapping optical coherence tomography, has been shown to extract parameters, such as capillary density and vessel diameter, and key clinical markers associated with early changes in microvascular diseases. However, OCT has limited spatial resolution in both the transverse and depth directions. Here, we extend this correlation mapping technique to other microscopy modalities, including confocal microscopy, and take advantage of the higher spatial resolution offered by these modalities. The technique is achieved as a processing step on microscopy images and does not require any modification to the microscope hardware. Results are presented which show that this correlation mapping microscopy technique can extend the capabilities of conventional microscopy to enable mapping of vascular networks in vivo with high spatial resolution in both the transverse and depth directions.

Macrophages adopt a variety of phenotypes that are a reflection of the many functions they perform as part of the immune system. In particular, metabolism is a phenotypic trait that differs between classically activated, proinflammatory macrophages, and alternatively activated, prohealing macrophages. Inflammatory macrophages have a metabolism based on glycolysis while alternatively activated macrophages generally rely on oxidative phosphorylation to generate chemical energy. We employ this shift in metabolism as an endogenous marker to identify the phenotype of individual macrophages via live-cell fluorescence lifetime imaging microscopy (FLIM). We demonstrate that polarized macrophages can be readily discriminated with the aid of a phasor approach to FLIM, which provides a fast and model-free method for analyzing fluorescence lifetime images.

Most ovarian cancers are diagnosed at advanced stages due to the lack of efficacious screening techniques. Photoacoustic tomography (PAT) has a potential to image tumor angiogenesis and detect early neovascular changes of the ovary. We have developed a coregistered PAT and ultrasound (US) prototype system for real-time assessment of ovarian masses. Features extracted from PAT and US angular beams, envelopes, and images were input to a logistic classifier and a support vector machine (SVM) classifier to diagnose ovaries as benign or malignant. A total of 25 excised ovaries of 15 patients were studied and the logistic and SVM classifiers achieved sensitivities of 70.4 and 87.7%, and specificities of 95.6 and 97.9%, respectively. Furthermore, the ovaries of two patients were noninvasively imaged using the PAT/US system before surgical excision. By using five significant features and the logistic classifier, 12 out of 14 images (86% sensitivity) from a malignant ovarian mass and all 17 images (100% specificity) from a benign mass were accurately classified; the SVM correctly classified 10 out of 14 malignant images (71% sensitivity) and all 17 benign images (100% specificity). These initial results demonstrate the clinical potential of the PAT/US technique for ovarian cancer diagnosis.

We present a generalized strategy for direct reconstruction in pharmacokinetic diffuse fluorescence tomography (DFT) with CT-analogous scanning mode, which can accomplish one-step reconstruction of the indocyanine-green pharmacokinetic-rate images within in vivo small animals by incorporating the compartmental kinetic model into an adaptive extended Kalman filtering scheme and using an instantaneous sampling dataset. This scheme, compared with the established indirect and direct methods, eliminates the interim error of the DFT inversion and relaxes the expensive requirement of the instrument for obtaining highly time-resolved date-sets of complete 360 deg projections. The scheme is validated by two-dimensional simulations for the two-compartment model and pilot phantom experiments for the one-compartment model, suggesting that the proposed method can estimate the compartmental concentrations and the pharmacokinetic-rates simultaneously with a fair quantitative and localization accuracy, and is well suitable for cost-effective and dense-sampling instrumentation based on the highly-sensitive photon counting technique.

The efficacy of existing therapies and the discovery of innovative treatments for central nervous system (CNS) diseases have been limited by the lack of appropriate methods to investigate complex molecular processes at the synaptic level. To improve our capability to investigate complex mechanisms of synaptic signaling and remodeling, we designed a fluorescence hyperspectral imaging platform to simultaneously track different subtypes of individual neurotransmitter receptors trafficking in and out of synapses. This imaging platform allows simultaneous image acquisition of at least five fluorescent markers in living neurons with a high-spatial resolution. We used quantum dots emitting at different wavelengths and functionalized to specifically bind to single receptors on the membrane of living neurons. The hyperspectral imaging platform enabled the simultaneous optical tracking of five different synaptic proteins, including subtypes of glutamate receptors (mGluR and AMPAR) and postsynaptic signaling proteins. It also permitted the quantification of their mobility after treatments with various pharmacological agents. This technique provides an efficient method to monitor several synaptic proteins at the same time, which could accelerate the screening of effective compounds for treatment of CNS disorders.

We demonstrate a fast two-color widefield fluorescence microendoscopy system capable of simultaneously detecting several disease targets in intact human ex vivo lung tissue. We characterize the system for light throughput from the excitation light emitting diodes, fluorescence collection efficiency, and chromatic focal shifts. We demonstrate the effectiveness of the instrument by imaging bacteria (Pseudomonas aeruginosa) in ex vivo human lung tissue. We describe a mechanism of bacterial detection through the fiber bundle that uses blinking effects of bacteria as they move in front of the fiber core providing detection of objects smaller than the fiber core and cladding (∼3  μm). This effectively increases the measured spatial resolution of 4  μm. We show simultaneous imaging of neutrophils, monocytes, and fungus (Aspergillus fumigatus) in ex vivo human lung tissue. The instrument has 10 nM and 50 nM sensitivity for fluorescein and Cy5 solutions, respectively. Lung tissue autofluorescence remains visible at up to 200 fps camera acquisition rate. The optical system lends itself to clinical translation due to high-fluorescence sensitivity, simplicity, and the ability to multiplex several pathological molecular imaging targets simultaneously.

Development of a block-based restoration (BBR) method that addresses spatially variant (SV) imaging in wide-field fluorescence microscopy of thick samples is presented. The BBR method is based on a block-based imaging model, which approximates SV imaging using an efficient orthonormal basis decomposition of multiple SV point-spread functions computed at block vertices. The effect of reducing the number of blocks needed to account for SV imaging on the restoration accuracy was investigated with simulations using a numerical lung tissue phantom relevant to biological studies. Results show that reducing the number of blocks by 82% and 98% resulted in a 19% and 27% reduction in restoration accuracy, respectively, thereby establishing a reasonable tradeoff between computational resources and accuracy. Comparison of the BBR method to existing methods (deconvolution) that do not account for SV imaging demonstrates a 90% improvement in restoration accuracy. BBR results from synthetic and experimental images of a controlled test sample with SV refractive index (RI) show consistency, providing a validation of the BBR approach. In this study, information from DIC and fluorescence images was combined to identify regions with changing RI within the imaging volume. The BBR method provides a first step toward computationally tractable reconstruction of images from thick samples.

The development and demonstration of a multiphoton photoacoustic imaging technique capable of providing high spatial resolution chemical images of subsurface tissue components as deep as 1.4 cm below the tissue surface is described. By combining multiphoton excitation in the diagnostic window (650 to 1100 nm), with ultrasonic detection of nonradiative relaxation events, it is possible to rapidly reconstruct three-dimensional, chemical specific, images of samples underneath overlying structures as well as chemical species of the same material. Demonstration of this technique for subsurface tissue differentiation is shown, with the ability to distinguish between grade III astrocytoma tissue and adjacent healthy tissue in blind studies. By employing photoacoustic signal detection, the high nonradiative relaxation rates of most biological tissue components (>90%) and the minimal signal attenuation of the resulting ultrasound compensate for excitation efficiency losses associated with two-photon absorption. Furthermore, the two-photon absorption process results in a highly localized excitation volume (ca., 60  μm). Characterization of the probing depth, spatial resolution, and ability to image through overlying structures is also demonstrated in this paper using tissue phantoms with well-characterized optical scattering properties, mimicking those of tissues.

Palmoplantar erythrodysesthesia (PPE), or hand-foot syndrome, is a cutaneous toxicity under various chemotherapeutics contributing to the most frequent side effects in patients treated with capecitabine (Xeloda®). The pathomechanism of PPE has been unclear. Here, the topical detection of capecitabine in the skin after oral application was shown in 10 patients receiving 2500  mg/m²/day capecitabine. Sweat samples were taken prior to and one week after oral administration of capecitabine. Using high-resolution continuum source absorption spectrometry, the changes in concentrations of fluorine, which is an ingredient of capecitabine, were quantified and statistically analyzed. Here, we show an increase in fluorine concentrations from 40±10  ppb (2±0.5  pM) before capecitabine administration to 27.7±11.8  ppm (14.6±6.5  nM) after application, p<0.001. The results show the secretion of capecitabine on the skin surface after oral administration, indicating a local toxic effect as a possible pathomechanism of PPE.

This study evaluated the influence of the irradiation with a short-pulse Er:YAG laser on the adhesion of composite resin to sound and eroded dentin (SD and ED). Forty-six samples of occlusal dentine, obtained from human molars, had half of their surface protected, while the other half was submitted to erosive cycles. Afterward, 23 samples were irradiated with Er:YAG laser, resulting in four experimental groups: SD, sound irradiated dentine (SID—Er:YAG, 50  μs, 2 Hz, 80 mJ, and 12.6  J/cm²), ED, and eroded irradiated dentin (EID—erosion + Er:YAG laser). A self-etching adhesive system was used, and then cylinders of composite resin were prepared. A microshear bond strength test was performed after 24 h storage (n=20). The morphology of SD and ED, with or without Er:YAG laser irradiation, was evaluated under scanning electron microscopy (n=3). Bond strength values (MPa) were subjected to analysis of variance followed by Tukey’s test. Statistically significant differences were found among the experimental groups: SD (9.76±3.39  B), SID (12.77±5.09 A), ED (5.12±1.72 D), and EID (7.62±3.39 C). Even though erosion reduces the adhesion to dentin, the surface irradiation with a short-pulse Er:YAG laser increases adhesion to both ED and SD.

This PDF file contains the errata for “JBO Vol. 21 Issue 04 Paper JBO-2016-0407-ERR” for JBO Vol. 21 Issue 04