JBO sustained its healthy growth in 2012. The number of manuscript submissions increased by 5% to 788. JBO’s success is attributed to the contributions of the authors, reviewers, guest editors, and editorial board members.

We present the use of correlation analysis on spectral data in order to quantify the amount of a given spectrum present with respect to a reference spectrum. The method is shown to be useful in analyzing hyperspectral fluorescence images. It is unhindered by the linear relationship assumed in linear spectral unmixing, and in addition, it is shown to be robust with respect to noise.

Cerenkov luminescence imaging is an emerging optical preclinical modality based on the detection of Cerenkov radiation induced by beta particles when traveling though biological tissues with a velocity greater than the speed of light. We present the first human Cerenkography obtained by detecting Cerenkov radiation escaping the thyroid gland of a patient treated for hyperthyroidism. The Cerenkov light was detected using an electron multiplied charge coupled device and a conventional C-mount lens. The system set-up has been tested by using a slab of ex vivo tissue equal to a 1 cm slice of chicken breast in order to simulate optical photons attenuation. We then imaged for 2 min the head and neck region of a patient treated orally 24 h before with 550 MBq of I-131. Co-registration between photographic and Cerenkov images showed a good localization of the Cerenkov light within the thyroid region. In conclusion, we showed that it is possible to obtain a planar image of Cerenkov photons escaping from a human tissue. Cerenkography is a potential novel medical tool to image superficial organs of patients treated with beta minus radiopharmaceuticals and can be extended to the imaging of beta plus emitters.

In recent years, an increasing effort has been devoted to the optimization of acquisition and reconstruction schemes for fluorescence molecular tomography (FMT). In particular, wide-field structured illumination and compression of the measured images have enabled significant reduction of the data set and, consequently, a decrease in both acquisition and processing times. FMT based on this concept has been recently demonstrated on a cylindrical phantom with a rotating-view scheme that significantly increases the reconstruction quality. In this work, we generalize the rotating-view scheme to arbitrary geometries and experimentally demonstrate its applicability to murine models. To the best of our knowledge this is the first time that FMT based on a rotating-view scheme with structured illumination and image compression has been applied to animals.

Photoacoustic microscopy (PAM) has achieved submicron lateral resolution in showing subcellular structures; however, relatively few endogenous subcellular contrasts have so far been imaged. Given that the hemeprotein, mostly cytochromes in general cells, is optically absorbing around the Soret peak (∼420  nm ), we implemented label-free PAM of cytochromes in cytoplasm for the first time. By measuring the photoacoustic spectra of the oxidized and reduced states of fibroblast lysate and fitting the difference spectrum with three types of cytochromes, we found that the three cytochromes account for more than half the optical absorption in the cell lysate at 420 nm wavelength. Fixed fibroblasts on slides were imaged by PAM at 422 and 250 nm wavelengths to reveal cytoplasms and nuclei, respectively, as confirmed by standard staining histology. PAM was also applied to label-free histology of mouse ear sections by showing cytoplasms and nuclei of various cells. PAM of cytochromes in cytoplasm is expected to be a high-throughput, label-free technique for studying live cell functions, which cannot be accomplished by conventional histology.

Ultrasound modulated optical tomography modulates scattered light within tissue by deterministically altering the optical properties of the sample with the ultrasonic pressure. This allows the light to be “tagged” and the degradation in spatial resolution associated with light scattering to be reduced. To our knowledge, this is the first demonstration of ultrasound modulated imaging of light generated within a scattering medium without an external light source. The technique has the potential to improve the spatial resolution of chemi- or bioluminescence imaging of tissue. Experimental results show that ultrasound modulated luminescence imaging can resolve two chemiluminescent objects separated by 5 mm at a 7 mm depth within a tissue phantom with a scattering coefficient of 30  cm −1 . The lateral resolution is estimated to be 3 mm. Monte Carlo simulations indicate that, with the current system signal to noise ratio, it is feasible to apply the approach to bioluminescence imaging when the concentration of bacteria in the animal organ is above 3.4×10 5 /μL .

The developing fetal brain is vulnerable to a variety of environmental agents including maternal ethanol consumption. Preclinical studies on the development and amelioration of fetal teratology would be significantly facilitated by the application of high resolution imaging technologies like optical coherence tomography (OCT) and high-frequency ultrasound (US). This study investigates the ability of these imaging technologies to measure the effects of maternal ethanol exposure on brain development, ex vivo, in fetal mice. Pregnant mice at gestational day 12.5 were administered ethanol (3  g/Kg b.wt.) or water by intragastric gavage, twice daily for three consecutive days. On gestational day 14.5, fetuses were collected and imaged. Three-dimensional images of the mice fetus brains were obtained by OCT and high-resolution US, and the volumes of the left and right ventricles of the brain were measured. Ethanol-exposed fetuses exhibited a statistically significant, 2-fold increase in average left and right ventricular volumes compared with the ventricular volume of control fetuses, with OCT-derived measures of 0.38 and 0.18  mm 3 , respectively, whereas the boundaries of the fetal mouse lateral ventricles were not clearly definable with US imaging. Our results indicate that OCT is a useful technology for assessing ventriculomegaly accompanying alcohol-induced developmental delay. This study clearly demonstrated advantages of using OCT for quantitative assessment of embryonic development compared with US imaging.

Confocal micro-Raman spectroscopy is employed to study the diffusion process of dimethyl sulfoxide (DMSO) in porcine skin optical clearing. The variation of DMSO concentration with time at different depths of the skin was obtained and then the DMSO diffusion coefficient with the passive diffusion model was calculated. Results show that it has a significant difference at different depths of the skin. Also, the DMSO concentration with the depth at different times was obtained and the same method was used to find the change law of the DMSO diffusion coefficient. Results indicate that it also changes with the treatment time. The experimental results are consistent with the theoretical model in a previous study. The current results demonstrate that Raman spectroscopy has the ability to quantitatively monitor the process of optical clearing.

We report here on studies of reorientation of human red blood cells (RBCs) in an optical trap. We have measured the time required, t re , for the plane of the RBC entering the optical trap to undergo a 90-deg rotation to acquire an edge on orientation with respect to the beam direction. This has been studied as a function of laser power, P , at the trap center. The variation of t re with increasing P shows an initial sharp decrease followed by a much smaller rate of further decrease. We find that this experimentally measured variation is not in complete agreement with the variation predicted by a theoretical model where the RBC is treated as a perfectly rigid circular disk-like body. We argue that this deviation arises due to deformation of the RBC. We further reason that this feature is dominated by the elastic behavior of the RBC membrane. We compare the studies carried out on normal RBCs with RBCs where varying conditions of membrane stiffness are expected. We propose that the value of energy used for maximum deformation possible during a reorientation process is an indicator of the membrane elasticity of the system under study.

The epigenetic control of heterochromatin deposition is achieved through a network of protein interactions mediated by the heterochromatin protein 1 (HP1). In earlier studies, we showed that the CCAAT/enhancer-binding protein alpha (C/EBPα), a transcription factor that controls cell differentiation, localizes to heterochromatin, and interacts with HP1α. Here, deletion and mutagenesis are combined with live-cell imaging approaches to characterize these protein interactions. The results demonstrate that the basic region and leucine zipper (BZip) domain of C/EBPα is sufficient for the interaction with HP1α in regions of heterochromatin. Fluorescence correlation spectroscopy and cross-correlation (FCS and FCCS) revealed very different diffusion profiles for HP1α and the BZip protein, and co-expression studies indicated that the mobile fractions of these nuclear proteins diffuse independently of one another. The steady-state interactions of these proteins in regions of heterochromatin were monitored using Förster resonance energy transfer (FRET). A point mutation in HP1α, W174A, which disrupts the interactions with proteins containing the common PxVxL motif did not affect the interaction with the BZip protein. In contrast, the HP1α W41A mutation, which prevents binding to methylated histones, exhibited greatly reduced FRET efficiency when compared to the wild type HP1α or HP1αW174A. The functional significance of these interactions is discussed.

Work remains to be done to understand the origins of ocular aberrations. We analyze lens aberrations of several species with Y-suture branches (bovine, ovine, and porcine) and their relationship with suture distribution. Aberrations are measured in vitro with a point diffraction interferometer in 10 different eyes of each species. The minimum number of Zernike polynomials minimizing the root mean square error of the wavefront is estimated by processing the interferograms. Through this we find significant amounts of astigmatism, coma, spherical aberration, and trefoil in the lenses of the three species. Moreover, we observe a high degree of correlation between the orientation of the lens sutures and the axis of nonrotationally symmetric aberrations. Our results point to lens sutures as the histological origin of the most significant lens aberrations: astigmatism, coma, and trefoil, but we are unable to find a major suture governing all the axes.

The effects of strong scattering in tissue limit the depth to which light may be focused. However, it has been shown that scattering may be reduced utilizing adaptive optics with a focused ultrasound (US) beam guidestar. The optical signal traveling through the US beam waist is frequency shifted and may be isolated with demodulation. This paper utilizes a multiphysics simulation to model the optical and US interactions in both synthetic tissue and random scattering media. The results illustrate that optical energy may be focused within a turbid medium utilizing a US guidestar. The results also suggest that optical energy travels preferentially along optical channels within a turbid medium.

In order to understand the so-called “social brain,” we need to monitor social interactions in face-to-face paradigms. Near-infrared spectroscopy (NIRS) is a promising technique to achieve this goal. We investigate the neuronal underpinnings of sharing a task in a proper social context. We record cortical activity by means of NIRS, while participants perform a joint Simon task. Different from other hemodynamic techniques, NIRS allows us to have both participants sit comfortably close to each other in a realistic and ecological environment. We found higher activation in the sensorimotor cortex while processing compatible trials as compared to incompatible ones referring to one’s own action alternative. Strikingly, when the participant was not responding because it was the turn of the other member of the pair, the inferior parietal was activated. This study provides twofold findings: first, they suggest that the joint Simon effect relies more on shared attentional mechanisms than a proper mapping of the other’s motor response. Second, they highlight the invaluable contribution NIRS can afford to social neuroscience in order to preserve ecological and naturalistic settings.

Solid optical tissue phantoms (OTPs) have been widely used for many purposes. This study introduces a spin-coating method (SCM) to fabricate a thin-layer solid OTP (TSOTP) with epidermal thickness. TSOTPs are fabricated by controlling the spin speed (250 to 2500 rpm), absorber concentration (0.2% to 1.0%), and the number of layers. The results show that the thicknesses of the TSOTPs are homogeneous in the region of interest. The one-layer TSOTP achieves maximum and minimum thicknesses of 65±0.28  μm (250 rpm) and 5.1±0.17  μm (2500 rpm), respectively, decreasing exponentially as a function of the spin speed. The thicknesses of the multilayer TSOTPs increases as a function of the number of layers and are correlated strongly with the spin speed (R 2 ≥0.95 ). The concentration of the OTP mixture does not directly affect the thickness of the TSOTP; however, the absorption coefficients exponentially increase as a function of absorber concentration (R 2 ≥0.98 ). These results suggest that the SCM can be used to fabricate homogeneous TSOTPs with various thicknesses by controlling the spin speed and number of layers. Finally, a double-layer OTP that combines epidermal TSOTP and dermal OTP is manufactured as a preliminary study to investigate the practical feasibility of TSOTPs.

The authors demonstrate in vivo dynamic process imaging using a label-free real-time optical-resolution photoacoustic microscope (OR-PAM). This reflection-mode system takes advantage of a 532-nm fiber laser source with a high pulse repetition rate of up to 600 kHz combined with a fast-scanning mirror system. Microvasculature in SCID mouse ears is imaged at near real-time (0.5 fps) for a 1×1  mm 2 field of view (FOV) with micron-scale lateral resolution. We also demonstrate imaging of cardiac-induced microhemodynamics in murine microvasculature at real-time frame-rates (30 fps) over a 250×250  μm 2 FOV using real-time C-scan OR-PAM with ability to provide sustained imaging with near real-time feedback for focusing and positioning.

In this report, we describe how to highly optimize a computer unified device architecture based platform to perform real-time processing of optical coherence tomography interferometric data and three-dimensional (3-D) volumetric rendering using a commercially available, cost-effective, graphics processing unit (GPU). The maximum complete attainable axial scan processing rate, including memory transfer and displaying B-scan frame, was 2.24 MHz for 16 bits pixel depth and 2048 fast Fourier transform size; the maximum 3-D volumetric rendering rate, including B-scan, en face view display, and 3-D rendering, was ∼23  volumes/second (volume size: 1024×256×200). To the best of our knowledge, this is the fastest processing rate reported to date with a single-chip GPU and the first implementation of real-time video-rate volumetric optical coherence tomography (OCT) processing and rendering that is capable of matching the acquisition rates of ultrahigh-speed OCT.

A novel photoacoustic thermometric method is presented for simultaneously imaging cells and sensing their temperature. With three-seconds-per-frame imaging speed, a temperature resolution of 0.2°C was achieved in a photo-thermal cell heating experiment. Compared to other approaches, the photoacoustic thermometric method has the advantage of not requiring custom-developed temperature-sensitive biosensors. This feature should facilitate the conversion of single-cell thermometry into a routine lab tool and make it accessible to a much broader biological research community.

We report a semiautomatic algorithm that is specialized for rapid analysis of beat-to-beat contraction-relaxation parameters of the heart in Drosophila. The presented algorithm adapts the general graph theoretical image segmentation algorithm and a histogram-based thresholding algorithm, which can measure many cardiac parameters, including heart rate, heart period, diastolic and systolic intervals, and end-diastolic and end-systolic areas. Additionally, dynamic cardiac functions, such as arrhythmia index and percent fractional shortening, can be automatically calculated for all the recorded heartbeats over significant periods of time.

Skeletal muscle pathologies cause irregularities in the normally periodic organization of the myofibrils. Objective grading of muscle morphology is necessary to assess muscle health, compare biopsies, and evaluate treatments and the evolution of disease. To facilitate such quantitation, we have developed a fast, sensitive, automatic imaging analysis software. It detects major and minor morphological changes by combining texture features and Fourier transform (FT) techniques. We apply this tool to second harmonic generation (SHG) images of muscle fibers which visualize the repeating myosin bands. Texture features are then calculated by using a Haralick gray-level cooccurrence matrix in MATLAB. Two scores are retrieved from the texture correlation plot by using FT and curve-fitting methods. The sensitivity of the technique was tested on SHG images of human adult and infant muscle biopsies and of mouse muscle samples. The scores are strongly correlated to muscle fiber condition. We named the software MARS (muscle assessment and rating scores). It is executed automatically and is highly sensitive even to subtle defects. We propose MARS as a powerful and unbiased tool to assess muscle health.

We present a method to automatically segment red blood cells (RBCs) visualized by digital holographic microscopy (DHM), which is based on the marker-controlled watershed algorithm. Quantitative phase images of RBCs can be obtained by using off-axis DHM along to provide some important information about each RBC, including size, shape, volume, hemoglobin content, etc. The most important process of segmentation based on marker-controlled watershed is to perform an accurate localization of internal and external markers. Here, we first obtain the binary image via Otsu algorithm. Then, we apply morphological operations to the binary image to get the internal markers. We then apply the distance transform algorithm combined with the watershed algorithm to generate external markers based on internal markers. Finally, combining the internal and external markers, we modify the original gradient image and apply the watershed algorithm. By appropriately identifying the internal and external markers, the problems of oversegmentation and undersegmentation are avoided. Furthermore, the internal and external parts of the RBCs phase image can also be segmented by using the marker-controlled watershed combined with our method, which can identify the internal and external markers appropriately. Our experimental results show that the proposed method achieves good performance in terms of segmenting RBCs and could thus be helpful when combined with an automated classification of RBCs.

A modular video endoscope is developed and tested to allow imaging in different modalities. This system incorporates white light imaging (WLI), cross-polarized imaging (CPI), and vital-dye fluorescence imaging (VFI), using interchangeable filter modules. CPI and VFI are novel endoscopic modalities that probe mucosal features associated with Barrett’s neoplasia. CPI enhances vasculature, while VFI enhances glandular architecture. In this pilot study, we demonstrate the integration of these modalities by imaging areas of Barrett’s metaplasia and neoplasia in an esophagectomy specimen. We verify that those key image features are also observed during an in vivo surveillance procedure. CPI images demonstrate improved visualization of branching blood vessels associated with neoplasia. VFI images show glandular architecture with increased glandular effacement associated with neoplasia. Results suggests that important pathologic features seen in CPI and VFI are not visible during standard endoscopic white light imaging, and thus the modalities may be useful in future in vivo studies for discriminating neoplasia from Barrett’s metaplasia. We further demonstrate that the integrated WLI/CPI/VFI endoscope is compatible with complementary high-resolution endomicroscopy techniques such as the high-resolution microendoscope, potentially enabling two-step (“red-flag” widefield plus confirmatory high-resolution imaging) protocols to be enhanced.

We present a novel, high-speed, polarization-sensitive, optical coherence tomography set-up for retinal imaging operating at a central wavelength of 1060 nm which was tested for in vivo imaging in healthy human volunteers. We use the system in combination with a Fourier domain mode locked laser with active spectral shaping which enables the use of forward and backward sweep in order to double the imaging speed without a buffering stage. With this approach and with a custom designed data acquisition system, we show polarization-sensitive imaging with an A-scan rate of 350 kHz. The acquired three-dimensional data sets of healthy human volunteers show different polarization characteristics in the eye, such as depolarization in the retinal pigment epithelium and birefringence in retinal nerve fiber layer and sclera. The increased speed allows imaging of large volumes with reduced motion artifacts. Moreover, averaging several two-dimensional frames allows the generation of high-definition B-scans without the use of an eye-tracking system. The increased penetration depth of the system, which is caused by the longer probing beam wavelength, is beneficial for imaging choroidal and scleral structures and allows automated segmentation of these layers based on their polarization characteristics.

We demonstrate the use of task-based image-quality metrics to compare various photoacoustic image-reconstruction algorithms, including a method based on the pseudoinverse of the system matrix, simple backprojection, filtered backprojection, and a method based on the Fourier transform. We use a three-dimensional forward model with a linear transducer array to simulate a photoacoustic imaging system. The reconstructed images correspond with two-dimensional slices of the object and are 128×128  pixels . In order to compare the algorithms, we use channelized Hotelling observers that predict the detection ability of human observers. We use two sets of channels: constant Q and difference of Gaussian spatial frequency channels. We look at three tasks, identification of a point source in a uniform background, identification of a 0.5-mm cube in a uniform background, and identification of a point source in a lumpy background. For the lumpy background task, which is the most realistic of the tasks, the method based on the pseudoinverse performs best according to both sets of channels.

We developed a new, easy, and objective method to detect gastric cancer using hyperspectral imaging (HSI) technology combining spectroscopy and imaging A total of 16 gastroduodenal tumors removed by endoscopic resection or surgery from 14 patients at Yamaguchi University Hospital, Japan, were recorded using a hyperspectral camera (HSC) equipped with HSI technology Corrected spectral reflectance was obtained from 10 samples of normal mucosa and 10 samples of tumors for each case The 16 cases were divided into eight training cases (160 training samples) and eight test cases (160 test samples) We established a diagnostic algorithm with training samples and evaluated it with test samples Diagnostic capability of the algorithm for each tumor was validated, and enhancement of tumors by image processing using the HSC was evaluated The diagnostic algorithm used the 726-nm wavelength, with a cutoff point established from training samples The sensitivity, specificity, and accuracy rates of the algorithm’s diagnostic capability in the test samples were 78.8% (63/80), 92.5% (74/80), and 85.6% (137/160), respectively Tumors in HSC images of 13 (81.3%) cases were well enhanced by image processing Differences in spectral reflectance between tumors and normal mucosa suggested that tumors can be clearly distinguished from background mucosa with HSI technology.

For clinical optoacoustic imaging, linear probes are preferably used because they allow versatile imaging of the human body with real-time display and free-hand probe guidance. The two-dimensional (2-D) optoacoustic image obtained with this type of probe is generally interpreted as a 2-D cross-section of the tissue just as is common in echo ultrasound. We demonstrate in three-dimensional simulations, phantom experiments, and in vivo mouse experiments that for vascular imaging this interpretation is often inaccurate. The cylindrical blood vessels emit anisotropic acoustic transients, which can be sensitively detected only if the direction of acoustic radiation coincides with the probe aperture. Our results reveal for this reason that the signal amplitude of different blood vessels may differ even if the vessels have the same diameter and initial pressure distribution but different orientation relative to the imaging plane. This has important implications for the image interpretation, for the probe guidance technique, and especially in cases when a quantitative reconstruction of the optical tissue properties is required.

Third-harmonic generation (THG) microscopy has been reported to provide intrinsic contrast in elastic fibers, cytoplasmic membrane, nucleus, actin filaments, lipid bodies, hemoglobin, and melanin in human skin. For advanced molecular imaging, exogenous contrast agents are developed for a higher structural or molecular specificity. We demonstrate the potential of the commonly adopted tattoo dye as a THG contrast agent for in vivo optical biopsy of human skin. Spectroscopy and microscopy experiments were performed on cultured cells with tattoo dyes, in tattooed mouse skin, and in tattooed human skin to demonstrate the THG enhancement effect. Compared with other absorbing dyes or nanoparticles used as exogenous THG contrast agents, tattoo dyes are widely adopted in human skin so that future clinical biocompatibility evaluation is relatively achievable. Combined with the demonstrated THG enhancement effect, tattoo dyes show their promise for future clinical imaging applications.

As a high-sensitivity imaging modality, bioluminescence tomography can reconstruct the three-dimensional (3-D) location of an internal luminescent source based on the 3-D surface light distribution. However, we can only get the multi-orientation two-dimensional (2-D) bioluminescence distribution in the experiments. Therefore, developing an accurate universal registration method is essential for following bioluminescent source reconstruction. We can then map the multi-orientation 2-D bioluminescence distribution to the 3-D surface derived from anatomical information with it. We propose a 2-D -to-3-D registration method based on iterated optimal projection and applied it in a registration and reconstruction study of three transgenic mice. Compared with traditional registration methods based on the fixed points, our method was independent of the markers and the registration accuracy of the three experiments was improved by 0.3, 0.5, and 0.4 pixels, respectively. In addition, based on the above two registration results using the two registration methods, we reconstructed the 3-D location of the inner bioluminescent source in the three transgenic mice. The reconstruction results showed that the average error distance between the center of the reconstructed element and the center of the real element were reduced by 0.32, 0.48, and 0.39 mm, respectively.

The main limitation of optical imaging techniques for studying biological tissues is light scattering leading to decreasing of transmittance, which lowers the imaging quality. In this case, an immersion method for optical clearing of biological tissues can provide a possible solution to this problem, because the application of biocompatible clearing agents can reduce light scattering. Optical clearing represents a promising approach to increasing the imaging depth for various techniques, for example, various spectroscopy and fluorescent methods, and optical coherence tomography (OCT). We investigate the improvement of light penetration depth in blood after application of polyethylene glycol, polypropylene glycol, propylene glycol, and hemoglobin solutions using an OCT system. Influence of clearing agents on light transport in tissues and blood was also investigated in the mouse tail vein.

We propose a new quantitative phase microscope based on spatial filtering of the beam carrying the sample-induced wavefront. A prototype built using a transmissive liquid crystal display for the experimental demonstration of the principle and preliminary results obtained with artificial and biological samples are presented.

Diffuse optical tomography (DOT) has been employed to derive spatial maps of physiologically important chromophores in the human breast, but the fidelity of these images is often compromised by boundary effects such as those due to the chest wall. We explore the image quality in fast, data-intensive analytic and algebraic linear DOT reconstructions of phantoms with subcentimeter target features and large absorptive regions mimicking the chest wall. Experiments demonstrate that the chest wall phantom can introduce severe image artifacts. We then show how these artifacts can be mitigated by exclusion of data affected by the chest wall. We also introduce and demonstrate a linear algebraic reconstruction method well suited for very large data sets in the presence of a chest wall.

An ultrahigh resolution spectral domain optical coherence tomography (SD-OCT) system is used to observe for the first time in vivo the early effect of sodium iodate (NaIO 3 ) toxicity on retinal morphology. Retinal degeneration is induced in rats via tail vein injection of NaIO 3 and structural changes in the outer retina are assessed longitudinally at baseline and 1, 2, 3, 6, 8, and 10 h, and 12 post drug administration with OCT, H&E histology, and IgG immunochemistry. Disruption of the structural integrity and changes in the optical reflectivity of the photoreceptor inner (IS) and outer segment (OS) layers are observed as early as 1 h post NaIO 3 injection. A new layer is observed in the OCT tomograms to form between the retinal pigmented epithelium and the photoreceptors OS a few hours post NaIO 3 injection. The dynamics and the low optical reflectivity of this layer, as well as cell swelling and disruption of the blood-retina barrier observed in the histological and immunohistochemistry cross-sections suggest that the layer corresponds to temporary fluid accumulation in the retina. Results from this study demonstrate the effectiveness of OCT technology for monitoring dynamic changes in the retinal morphology and provide better understanding of the early stages of outer retina degeneration induced by NaIO 3 toxicity.

Radiation-damaged nanodiamonds (DNDs) are potentially ideal optical contrast agents for photoacoustic (PA) imaging in biological tissues due to their low toxicity and high optical absorbance. PA imaging contrast agents have been limited to quantum dots and gold particles, since most existing carbon-based nanoparticles, including fluorescent nanodiamonds, do not have sufficient optical absorption in the near-infrared (NIR) range. A new DND by He + ion beam irradiation with very high NIR absorption was synthesized. These DNDs produced a 71-fold higher PA signal on a molar basis than similarly dimensioned gold nanorods, and 7.1 fmol of DNDs injected into rodents could be clearly imaged 3 mm below the skin surface with PA signal enhancement of 567% using an 820-nm laser wavelength.

Since conventional techniques using periodontal probes have inherent drawbacks in the diagnosis of different grades of gingival inflammation, development of noninvasive screening devices becomes significant. Diffuse reflectance (DR) spectra recorded with white light illumination is utilized to detect periodontal inflammation from the oxygenated hemoglobin absorption ratio R620/R575. A multispectral imaging system is utilized to record narrow-band DR images at 575 and 620 nm from the anterior sextant of the gingivia of 15 healthy volunteers and 25 patients (N=40 ). An experienced periodontist assesses the level of gingival inflammation at each site through periodontal probing and assigns diagnosis as healthy, mild, moderate, or severe inflammation. The DR image ratio R620/R575 computed for each pixel (8-µm resolution) from the monochrome images is pseudo-color-mapped to identify gingival inflammation sites. The DR image ratio values at each site are compared with clinical diagnosis to estimate the specificity and sensitivity of the DR imaging technique in inflammation mapping. The high diagnostic accuracy is utilized to detect underlying inflammation in six patients with a previous history of periodontitis.

We developed a stable, reproducible three-dimensional optical phantom for the evaluation of a wide-field endoscopic molecular imaging system. This phantom mimicked a human esophagus structure with flexibility to demonstrate body movements. At the same time, realistic visual appearance and diffuse spectral reflectance properties of the tissue were simulated by a color matching methodology. A photostable dye-in-polymer technology was applied to represent biomarker probed “hot-spot” locations. Furthermore, fluorescent target quantification of the phantom was demonstrated using a 1.2 mm ultrathin scanning fiber endoscope with concurrent fluorescence-reflectance imaging.

Hearing loss is the most common sensory deficit in the world, and most frequently it originates in the inner ear. Yet, the inner ear has been difficult to access for diagnosis because of its small size, delicate nature, complex three-dimensional anatomy, and encasement in the densest bone in the body. Evolving optical methods are promising to afford cellular diagnosis of pathologic changes in the inner ear. To appropriately interpret results from these emerging technologies, it is important to characterize optical properties of cochlear tissues. Here, we focus on that characterization using quantitative polarized light microscopy (qPLM) applied to unstained cochlear sections of the mouse, a common animal model of human hearing loss. We find that the most birefringent cochlear materials are collagen fibrils and myelin. Retardance of the otic capsule, the spiral ligament, and the basilar membrane are substantially higher than that of other cochlear structures. Retardance of the spiral ligament and the basilar membrane decrease from the cochlear base to the apex, compared with the more uniform retardance of other structures. The intricate structural details revealed by qPLM of unstained cochlear sections ex vivo strongly motivate future application of polarization-sensitive optical coherence tomography to human cochlea in vivo.

There is a growing body of literature showing the usefulness of multiphoton tomography (MPT) and fluorescence lifetime imaging for in situ characterization of skin constituents and the ensuing development of noninvasive diagnostic tools against skin diseases. Melanin and pigmentation-associated skin cancers constitute some of the major applications. We show that MPT and fluorescence lifetime imaging can be used to measure changes in cutaneous melanin concentration and that these can be related to the visible skin color. Melanin in the skin of African, Indian, Caucasian, and Asian volunteers is detected on the basis of its emission wavelength and fluorescence lifetimes in solution and in a melanocyte-keratinocyte cell culture. Fluorescence intensity is used to characterize the melanin content and distribution as a function of skin type and depth into the skin (stratum granulosum and stratum basale). The measured fluorescence intensities in given skin types agree with melanin amounts reported by others using biopsies. Our results suggest that spatial distribution of melanin in skin can be studied using MPT and fluorescence lifetime imaging, but further studies are needed to ascertain that the method can resolve melanin amount in smaller depth intervals.

The solution of the forward equation that models the transport of light through a highly scattering tissue material in diffuse optical tomography (DOT) using the finite element method gives flux density (Φ ) at the nodal points of the mesh. The experimentally measured flux (U measured ) on the boundary over a finite surface area in a DOT system has to be corrected to account for the system transfer functions (R) of various building blocks of the measurement system. We present two methods to compensate for the perturbations caused by R and estimate true flux density (Φ ) from U cal measured . In the first approach, the measurement data with a homogeneous phantom (U homo measured ) is used to calibrate the measurement system. The second scheme estimates the homogeneous phantom measurement using only the measurement from a heterogeneous phantom, thereby eliminating the necessity of a homogeneous phantom. This is done by statistically averaging the data (U hetero measured ) and redistributing it to the corresponding detector positions. The experiments carried out on tissue mimicking phantom with single and multiple inhomogeneities, human hand, and a pork tissue phantom demonstrate the robustness of the approach.

Accurate quantification of Förster resonance energy transfer (FRET) using intensity-based methods is difficult due to the overlap of fluorophore excitation and emission spectra. Consequently, mechanisms are required to remove bleedthrough of the donor emission into the acceptor channel and direct excitation of the acceptor when aiming to excite only the donor fluorophores. Methods to circumvent donor bleedthrough using the unmixing of emission spectra have been reported, but these require additional corrections to account for direct excitation of the acceptor. Here we present an alternative method for robust quantification of FRET efficiencies based upon the simultaneous spectral unmixing of both excitation and emission spectra. This has the benefit over existing methodologies in circumventing the issue of donor bleedthrough and acceptor cross excitation without the need for additional corrections. Furthermore, we show that it is applicable with as few as two excitation wavelengths and so can be used for quantifying FRET efficiency in microscope images as easily as for data collected on a spectrofluorometer. We demonstrate the accuracy of the approach by reproducing efficiency values in well characterized FRET standards: HEK cells expressing a variety of linked cerulean and venus fluorescent proteins. Finally we describe simple ImageJ plugins that can be used to calculate and create images of FRET efficiencies from microscope images.

A Cerenkov fiber-optic dosimeter (CFOD) is fabricated using plastic optical fibers to measure Cerenkov radiation induced by a therapeutic photon beam. We measured the Cerenkov radiation generated in optical fibers in various irradiation conditions to evaluate the usability of Cerenkov radiation for a photon beam therapy dosimetry. As a results, the spectral peak of Cerenkov radiation was measured at a wavelength of 515 nm, and the intensity of Cerenkov radiation increased linearly with increasing irradiated length of the optical fiber. Also, the intensity peak of Cerenkov radiation was measured in the irradiation angle range of 30 to 40 deg. In the results of Monte Carlo N-particle transport code simulations, the relationship between fluxes of electrons over Cerenkov threshold energy and energy deposition of a 6 MV photon beam had a nearly linear trend. Finally, percentage depth doses for the 6 MV photon beam could be obtained using the CFOD and the results were compared with those of an ionization chamber. Here, the mean dose difference was about 0.6%. It is anticipated that the novel and simple CFOD can be effectively used for measuring depth doses in radiotherapy dosimetry.

During extracorporeal circulation (ECC), blood is periodically sampled and analyzed to maintain the blood-gas status of the patient within acceptable limits. This protocol has well-known drawbacks that may be overcome by continuous monitoring. We present the characterization of a new pH sensor for continuous monitoring in ECC. This monitoring device includes a disposable fluorescence-sensing element directly in contact with the blood, whose fluorescence intensity is strictly related to the pH of the blood. In vitro experiments show no significant difference between the blood gas analyzer values and the sensor readings; after proper calibration, it gives a correlation of R>0.9887 , and measuring errors were lower than the 3% of the pH range of interest (RoI) with respect to a commercial blood gas analyzer. This performance has been confirmed also by simulating a moderate ipothermia condition, i.e., blood temperature 32°C, frequently used in cardiac surgery. In ex vivo experiments, performed with animal models, the sensor is continuously operated in an extracorporeal undiluted blood stream for a maximum of 11 h. It gives a correlation of R>0.9431 , and a measuring error lower than the 3% of the pH RoI with respect to laboratory techniques.

The capabilities of micro-Raman spectroscopy for differentiating normal and malignant nasopharyngeal tissues were evaluated. Raman scattering signals were acquired from 22 normal and 52 malignant nasopharyngeal tissue samples. Distinctive spectral differences in Raman spectra between normal and malignant nasopharyngeal tissues were found, particularly in the spectral ranges of 853, 937, 1094, 1209, 1268, 1290 to 1340, 1579, and 1660  cm −1 , which primarily contain signals related to proteins, DNA, and lipids. Compared to normal tissues, the band intensity located at 853, and 937  cm −1 were significantly lower for cancerous tissues (p<0.05 ), while the band intensity located at 1094, 1209, 1268, and 1579  cm −1 were significantly higher (p<0.05 ). The band intensity located at 1290 to 1340, and 1660  cm −1 were also higher for cancerous tissues; but the differences were not statistically significant (p>0.05 ). Principal component analysis (PCA) and linear discriminate analysis (LDA) were employed to generate diagnostic algorithms for classification of Raman spectra of the two nasopharyngeal tissue types. The PCA-LDA algorithms together with leave-one-out, cross-validation technique yielded diagnostic sensitivity of 92% and specificity of 82%. This work demonstrated that the Raman spectroscopy technique associated with PCA-LDA diagnostic algorithms has potential for improving the diagnosis of nasopharyngeal cancers.

Currently, photoplethysmography (PPG) is a frequently studied optical blood pulsation detection technique among biophotonic and biomedical researchers due to the fact that it shows high potential for estimating the arterial stiffness (AS). The extraction of diagnostically useful information requires standardized measurement procedure with good repeatability. However, the effects of a crucially important factor—the optimal contact pressure (CP) of the probe—are often ignored. Also, CP values are not reported to evaluate those effects. It is hypothesized that AS estimated from PPG pulse wave 2nd derivative parameter b/a is strongly inconsistent when recorded at nonoptimal probe CP. Our pilot study confirmed this during in vivo PPG recordings from conduit artery sites on five healthy subjects at variable probe CP (0 to 15 kPa) by using 880 nm reflectance type sensor, force transducer, and PPG alternating current (AC) signal pulse area derived optimal CP criterion. The b/a values, calculated from PPG with variable CP, showed variation >300  percent. In contrast, at the optimal CP, the b/a showed high repeatability (coefficient of variability <5  percent). The effect has been explained with exponential pulse pressure-volume relationship model which indicates the optimal CP range.

A noninvasive head probe that combines near-infrared spectroscopy (NIRS) and electroencephalography (EEG) for simultaneous measurement of neural dynamics and hemodynamics in the brain is presented. It is composed of a compliant expandable mechanism that accommodates a wide range of head size variation and an elastomeric web that maintains uniform sensor contact pressure on the scalp as the mechanism expands and contracts. The design is intended to help maximize optical and electrical coupling and to maintain stability during head movement. Positioning electrodes at the inion, nasion, central, and preauricular fiducial locations mechanically shapes the probe to place 64 NIRS optodes and 65 EEG electrodes following the 10–5 scalp coordinates. The placement accuracy, precision, and scalp pressure uniformity of the sensors are evaluated. A root-mean-squared (RMS) positional precision of 0.89±0.23  mm , percent arc subdivision RMS accuracy of 0.19±0.15% , and mean normal force on the scalp of 2.28±0.88  N at 5 mm displacement were found. Geometric measurements indicate that the probe will accommodate the full range of adult head sizes. The placement accuracy, precision, and uniformity of sensor contact pressure of the proposed head probe are important determinants of data quality in noninvasive brain monitoring with simultaneous NIRS-EEG.

Diffuse reflectance and fluorescence spectroscopy are used to detect histopathological abnormalities of an epileptic brain in a human subject study. Static diffuse reflectance and fluorescence spectra are acquired from normal and epileptic brain areas, defined by electrocorticography (ECoG), from pediatric patients undergoing epilepsy surgery. Biopsy specimens are taken from the investigated sites within an abnormal brain. Spectral analysis reveals significant differences in diffuse reflectance spectra and the ratio of fluorescence and diffuse reflectance spectra from normal and epileptic brain areas defined by ECoG and histology. Using these spectral differences, tissue classification models with accuracy above 80% are developed based on linear discriminant analysis. The differences between the diffuse reflectance spectra from the normal and epileptic brain areas observed in this study are attributed to alterations in the static hemodynamic characteristics of an epileptic brain, suggesting a unique association between the histopathological and the hemodynamic abnormalities in an epileptic brain.

Preterm infants are highly susceptible to ischemic brain injury; consequently, continuous bedside monitoring to detect ischemia before irreversible damage occurs would improve patient outcome. In addition to monitoring cerebral blood flow (CBF), assessing the cerebral metabolic rate of oxygen (CMRO 2 ) would be beneficial considering that metabolic thresholds can be used to evaluate tissue viability. The purpose of this study was to demonstrate that changes in absolute CMRO 2 could be measured by combining diffuse correlation spectroscopy (DCS) with time-resolved near-infrared spectroscopy (TR-NIRS). Absolute CBF was determined using bolus-tracking TR-NIRS to calibrate the DCS measurements. Cerebral venous blood oxygenation (SvO 2 ) was determined by multiwavelength TR-NIRS measurements, the accuracy of which was assessed by directly measuring the oxygenation of sagittal sinus blood. In eight newborn piglets, CMRO 2 was manipulated by varying the anesthetics and by injecting sodium cyanide. No significant differences were found between the two sets of SvO 2 measurements obtained by TR-NIRS or sagittal sinus blood samples and the corresponding CMRO 2 measurements. Bland–Altman analysis showed a mean CMRO 2 difference of 0.0268±0.8340  mL O 2 /100  g/min between the two techniques over a range from 0.3 to 4 mL O 2 /100  g/min .

The ability of combining serum surface-enhanced Raman spectroscopy (SERS) with support vector machine (SVM) for improving classification esophageal cancer patients from normal volunteers is investigated. Two groups of serum SERS spectra based on silver nanoparticles (AgNPs) are obtained: one group from patients with pathologically confirmed esophageal cancer (n=30 ) and the other group from healthy volunteers (n=31 ). Principal components analysis (PCA), conventional SVM (C-SVM) and conventional SVM combination with PCA (PCA-SVM) methods are implemented to classify the same spectral dataset. Results show that a diagnostic accuracy of 77.0% is acquired for PCA technique, while diagnostic accuracies of 83.6% and 85.2% are obtained for C-SVM and PCA-SVM methods based on radial basis functions (RBF) models. The results prove that RBF SVM models are superior to PCA algorithm in classification serum SERS spectra. The study demonstrates that serum SERS in combination with SVM technique has great potential to provide an effective and accurate diagnostic schema for noninvasive detection of esophageal cancer.

We present a novel way to recover intrinsic fluorescence in turbid media based on Monte Carlo generated look-up tables and making use of a diffuse reflectance measurement taken at the same location. The method has been validated on various phantoms with known intrinsic fluorescence and is benchmarked against photon-migration methods. This new method combines more flexibility in the probe design with fast reconstruction and showed similar reconstruction accuracy as found in other reconstruction methods.

Heparin is the most widely used anti-coagulant for the prevention of blood clots in patients undergoing certain types of surgeries including open heart surgeries and dialysis. The precise monitoring of heparin amount in patients’ blood is crucial for reducing the morbidity and mortality in surgical environments. Based upon these considerations, we have used Raman spectroscopy in conjunction with partial least squares (PLS) analysis to measure heparin concentration at clinical level which is less than 10 United States Pharmacopeia (USP) in serum. The PLS calibration model was constructed from the Raman spectra of different concentrations of heparin in serum. It showed a high coefficient of determination (R 2 >0.91 ) between the spectral data and heparin level in serum along with a low root mean square error of prediction ∼4  USP/ml . It enabled the detection of extremely low concentrations of heparin in serum (∼8  USP/ml ) as desirable in clinical environment. The proposed optical method has the potential of being implemented as the point-of-care testing procedure during surgeries, where the interest is to rapidly monitor low concentrations of heparin in patient’s blood.

Results of the structural analysis of urinary sediments by means of infrared spectral microscopy are presented. The results are in good agreement with the results of standard optical microscopy in the case of single-component and crystalline urinary sediments. It is found that for noncrystalline or multicomponent sediments, the suggested spectroscopic method is superior to optical microscopy. The chemical structure of sediments of any molecular origin can be elucidated by this spectroscopic method. The method is sensitive enough to identify solid particles of drugs present in urine. Sulfamethoxazole and traces of other medicines are revealed in this study among the other sediments. We also show that a rather good correlation exists between the type of urinary sediments and the renal stones removed from the same patient. Spectroscopic studies of urinary stones and corresponding sediments from 76 patients suffering from renal stone disease reveal that in 73% of cases such correlation exists. This finding is a strong argument for the use of infrared spectral microscopy to prevent kidney stone disease because stones can be found in an early stage of formation by using the nonintrusive spectroscopic investigation of urinary sediments. Some medical recommendations concerning the overdosing of certain pharmaceuticals can also be derived from the spectroscopic studies of urinary sediments.

Several optical techniques and fiber-optic probe systems have been designed to measure the optical properties of tissue. While a wide range of options is often beneficial, it poses a problem to investigators selecting which method to use for their biomedical application of interest. We present a methodology to optimally select a probe that matches the application requirements. Our method is based both on matching a probe’s mean sampling depth with the optimal diagnostic depth of the clinical application and on choosing a probe whose interrogation depth and path length is the least sensitive to alterations in the target medium’s optical properties. Satisfying these requirements ensures that the selected probe consistently assesses the relevant tissue volume with minimum variability. To aid in probe selection, we have developed a publicly available graphical user interface that takes the desired sampling depth and optical properties of the medium as its inputs and automatically ranks different techniques in their ability to robustly target the desired depth. Techniques investigated include single fiber spectroscopy, differential path length spectroscopy, polarization-gating, elastic light scattering spectroscopy, and diffuse reflectance. The software has been applied to biological case studies.

Fiber-optic attraction of urinary stones during laser lithotripsy may be exploited to manipulate stone fragments inside the urinary tract without mechanical grasping tools, saving the urologist time and space in the ureteroscope working channel. We compare thulium fiber laser (TFL) high pulse rate/low pulse energy operation to conventional holmium:YAG low pulse rate/high pulse energy operation for fiber-optic suctioning of plaster-of-paris (PoP) stone phantoms. A TFL (wavelength of 1908 nm, pulse energy of 35 mJ, pulse duration of 500 μs, and pulse rate of 10 to 350 Hz) and a holmium laser (wavelength of 2120 nm, pulse energy of 35 to 360 mJ, pulse duration of 300 μs, and pulse rate of 20 Hz) were tested using 270-μm-core optical fibers. A peak drag speed of ∼2.5  mm/s was measured for both TFL (35 mJ and 150 to 250 Hz) and holmium laser (210 mJ and 20 Hz). Particle image velocimetry and thermal imaging were used to track water flow for all parameters. Fiber-optic suctioning of urinary stone phantoms is feasible. TFL operation at high pulse rates/low pulse energies is preferable to holmium operation at low pulse rates/high pulse energies for rapid and smooth stone pulling. With further development, this novel technique may be useful for manipulating stone fragments in the urinary tract.

This article [J. Biomed. Opt.. 18, , 026014 (2013)] was originally published online on 6 February 2013 with an error in the title: the word “studied” was added. The corrected title appears above. This article was corrected online on 13 February 2013. It appears correctly in print. The Authors

This article [J. Biomed. Opt.. 18, , 026022 (2013)] was originally published online on 14 February 2013 with an error in Fig. 7 caption. The word “corneum” has been replaced by “granulosum.” The corrected caption reads: Fig. 7 Maximum area-normalized fluorescence intensity corresponding to the lifetime τ 1 in stratum granulosum and stratum basale versus mean L ∗ value (Table 4) in (a) dorsal stratum granulosum; (b) dorsal stratum basale; (c) volar stratum granulosum; and (d) volar stratum basale. Data (mean±s:e:m: ) from the five volunteers. This article was corrected online on 20 February 2013. It appears correctly in print. The Authors

The second edition of Fundamentals of Light Microscopy and Electronic Imaging is a collaborative effort of Douglas B. Murphy and Michael W. Davidson. Murphy is known for his establishment of the School of Medicine Microscope Facility at the Johns Hopkins School of Medicine. He directed the facility until 2006 and has superb experience as a teacher of microscopy, optics, and image processing. He was the sole author of the first edition of this text and reference book. Davidson, who is at the National High Magnetic Field Laboratory, The Florida State University, is well known for his development of websites for online learning of microscopy; especially his development of dynamic Java and Flash tutorials to illustrate the principles and the applications. Davidson and his collaborators have produced very useful dynamic Java and Flash tutorials which are highly recommended. Nikon supports the site: CrossRef[[XSLOpenURL/]]. Olympus supports the Microscopy Resource Center at CrossRef[[XSLOpenURL/]]. I have used both of these education sites, and I highly recommend them to the readers of the Journal of Biomedical Optics. The combined work of these two authors results in a recommended textbook and reference that is an excellent starting point for someone new to the field of microscopy. While there is much information contained within the covers, from theory to applications, to demonstrations that the reader can perform in the laboratory, to discussions of limitations, it must be stated that this book is directed to the novice in microscopy and is an entry-level exposition of the fundamentals of the subject, devoid of the mathematics that is usual for optics books. With that disclaimer, I think that this highly acclaimed book is well-suited to the person who is attempting to learn the fundamentals of microscopy and electronic imaging.