This PDF file contains the editorial “JBO 2014 List of Reviewers” for JBO Vol. 20 Issue 01

We report a novel fluorescence imaging approach to imaging nonfluorescence-labeled biological tissue samples. The method was demonstrated by imaging neurons in Golgi-Cox-stained and epoxy-resin-embedded samples through the excitation of the background fluorescence of the specimens. The dark neurons stood out clearly against background fluorescence in the images, enabling the tracing of a single dendritic spine using both confocal and wide-field fluorescence microscopy. The results suggest that the reported fluorescence imaging method would provide an effective alternative solution to image nonfluorescence-labeled samples, and it allows tracing the dendritic spine structure of neurons.

Deep vein thrombosis (DVT) has become a severe disease with a rising incidence rate. The conventional diagnosis relies on complicated imaging modalities that may also involve invasive contrast agent injection and ionizing procedures (e.g., venography). Noninvasive near-infrared spectroscopy (NIRS) methods have been explored which required the DVT patients to follow some exercise protocols. Here, we attempt to use portable NIRS under patients’ natural state for DVT diagnosis. Nine DVT patients and seven healthy subjects participated in NIRS measurements of concentration of oxy- and deoxy-hemoglobins (Δ[HbO₂] and Δ[Hb]) relative to data on a tissue mimicking phantom at six particular sites of calves. It was found that Δ[HbO₂] is significantly lower in DVT patients than healthy ones, whereas Δ[Hb]) is distinctly higher. Moreover, after thrombolytic therapy, both Δ[HbO₂] and Δ[Hb] in DVT calves assume a gradual convergence to the curves of healthy ones. This reveals the potential of NIRS for the noninvasive, continuous, and straightforward monitoring/therapeutic effect evaluation of DVT in clinics with appropriate bedside monitoring capability.

There is a need for accurate, high-throughput, functional measures to gauge the efficacy of potential drugs in living cells. As an early marker of drug response in cells, cellular metabolism provides an attractive platform for high-throughput drug testing. Optical techniques can noninvasively monitor NADH and FAD, two autofluorescent metabolic coenzymes. The autofluorescent redox ratio, defined as the autofluorescence intensity of NADH divided by that of FAD, quantifies relative rates of cellular glycolysis and oxidative phosphorylation. However, current microscopy methods for redox ratio quantification are time-intensive and low-throughput, limiting their practicality in drug screening. Alternatively, high-throughput commercial microplate readers quickly measure fluorescence intensities for hundreds of wells. This study found that a commercial microplate reader can differentiate the receptor status of breast cancer cell lines (p<0.05) based on redox ratio measurements without extrinsic contrast agents. Furthermore, microplate reader redox ratio measurements resolve response (p<0.05) and lack of response (p>0.05) in cell lines that are responsive and nonresponsive, respectively, to the breast cancer drug trastuzumab. These studies indicate that the microplate readers can be used to measure the redox ratio in a high-throughput manner and are sensitive enough to detect differences in cellular metabolism that are consistent with microscopy results.

The issues involved with recording vital functions in the magnetic resonance imaging (MRI) environment using fiber-optic sensors are considered in this paper. Basic physiological parameters, such as respiration and heart rate, are fundamental for predicting the risk of anxiety, panic, and claustrophobic episodes in patients undergoing MRI examinations. Electronic transducers are generally hazardous to the patient and are prone to erroneous operation in heavily electromagnetically penetrated MRI environments; however, nonmetallic fiber-optic sensors are inherently immune to electromagnetic effects and will be crucial for acquiring the above-mentioned physiological parameters. Forty-seven MRI-tested or potentially MRI-compatible sensors have appeared in the literature over the last 20 years. The author classifies these sensors into several categories and subcategories, depending on the sensing element placement, method of application, and measurand type. The author includes five in-house-designed fiber Bragg grating based sensors and shares experience in acquiring physiological measurements during MRI scans. This paper aims to systematize the knowledge of fiber-optic techniques for recording life functions and to indicate the current directions of development in this area.

We created a simulation model to investigate the characteristics of fluorescence in two-photon-excited samples. In the model, the sample is a diffusible solution of fluorophore molecules, which is divided into cubic cells and illuminated by a train of focused laser pulses described as a Gaussian beam. Simulating the state transitions according to a multilevel photodynamic model (also including photobleaching and intersystem crossing), the simulator provides the expected number and the spatial distribution of emitted photons over time. Our simulations demonstrated how the illumination laser power, diffusion, and the photodynamic parameters of the fluorophore affect fluorescence. We revealed the unusual fluorescent profile that evolves as photobleaching progresses: the most photons are not emitted from the focus (where a “dark hole” appears) but from an ellipsoid around the focus. The model could be adapted to several fluorescent techniques (such as two-photon microscopy and fluorescence recovery after photobleaching). Furthermore, it might help to optimize the operating parameters of the measurement devices (e.g., in order to reach higher image quality and lower photobleaching).

In three-dimensional (3-D) modeling of light transport in heterogeneous biological structures using the Monte Carlo (MC) approach, space is commonly discretized into optically homogeneous voxels by a rectangular spatial grid. Any round or oblique boundaries between neighboring tissues thus become serrated, which raises legitimate concerns about the realism of modeling results with regard to reflection and refraction of light on such boundaries. We analyze the related effects by systematic comparison with an augmented 3-D MC code, in which analytically defined tissue boundaries are treated in a rigorous manner. At specific locations within our test geometries, energy deposition predicted by the two models can vary by 10%. Even highly relevant integral quantities, such as linear density of the energy absorbed by modeled blood vessels, differ by up to 30%. Most notably, the values predicted by the customary model vary strongly and quite erratically with the spatial discretization step and upon minor repositioning of the computational grid. Meanwhile, the augmented model shows no such unphysical behavior. Artifacts of the former approach do not converge toward zero with ever finer spatial discretization, confirming that it suffers from inherent deficiencies due to inaccurate treatment of reflection and refraction at round tissue boundaries.

The damage mechanism for near-infrared radiation (IRR) induced cataract is unclear. Both a photochemical and a thermal mechanism were suggested. The current paper aims to elucidate a photochemical effect based on investigation of irradiance-exposure time reciprocity. Groups of 20 rats were unilaterally exposed to 96-W/cm² IRR at 1090 nm within the dilated pupil accumulating 57, 103, 198, and 344  kJ/cm2, respectively. Temperature was recorded at the limbus of the exposed eye. Seven days after exposure, the lenses were macroscopically imaged and light scattering was quantitatively measured. The average maximum temperature increases for exposure times of 10, 18, 33, and 60 min were expressed as 7.0±1.1, 6.8±1.1, 7.6±1.3, and 7.4±1.1°C [CI (0.95)] at the limbus of the exposed eye. The difference of light scattering in the lenses between exposed and contralateral not-exposed eyes was 0.00±0.02, 0.01±0.03, −0.01±0.02, and −0.01±0.03 transformed equivalent diazepam concentration (tEDC), respectively, and no apparent morphological changes in the lens were observed. An exposure to 96-W/cm² 1090-nm IRR projected on the cornea within the dilated pupil accumulating radiant exposures up to 344  kJ/cm² does not induce cataract if the temperature rise at the limbus is <8°C. This is consistent with a thermal damage mechanism for IRR-induced cataract.

Nitric oxide (NO) has key regulatory roles in various biological and medical processes. The control of its local concentration, which is crucial for obtaining the desired effect, can be achieved with exogenous NO donors. Release of NO from metal-nitrosyl complexes upon exposure to light is a strategy that could allow for the site-specific delivery of the reactive species NO to physiological targets. The photodissociation of NO from two nitrosylruthenium(II) isomer complexes {cis- and trans-[Ru(OAc)(2mqn)₂NO]} was demonstrated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry spectra, and electron paramagnetic resonance spectra further prove the photoinduced NO release by spin trapping of NO free radicals upon photoirradiation. Real-time NO release was quantitatively measured by electrochemistry with an NO-specific electrode. The quantitative control of NO release from [Ru(OAc)(2mqn)₂NO] in aqueous solutions was done by photoirradiation at different wavelengths. Both isomers show photoinduced damage on plasmid DNA, but the trans isomer has higher cytotoxicity and photocytotoxicity activity against the HeLa tumor cell line than that of the cis isomer. Nitrosylruthenium(II) complex, with 8-quinolinol derivatives as ligands, has a great potential as a photoactivated NO donor reagent for biomedical applications.

The observed color of an object is influenced by the spectral distribution of an illuminant impinging upon it. Here we explored a method to obtain optimal illumination spectra for local contrast enhancement based on human vision. First, multispectral imaging was used to measure the spectral reflectance of the sample and color segmentation was used to extract its color features. Then we obtained the target-specific optimal illumination by maximizing the color differences of mutual colors in our sample tissue. To verify the effectiveness of this method, simulated images under the optimized illumination were compared to illumination with the standard illuminant D65 and a cool white light-emitting diode (5500 K). Results showed that the sample under the optimized illumination had a better perceptual color contrast.

Investigating the elasticity of ocular tissue (cornea and intraocular lens) could help the understanding and management of pathologies related to biomechanical deficiency. In previous studies, we introduced a setup based on optical coherence tomography for shear wave elastography (SWE) with high resolution and high sensitivity. SWE determines tissue stiffness from the propagation speed of shear waves launched within tissue. We proposed acoustic radiation force to remotely induce shear waves by focusing an ultrasound (US) beam in tissue, similar to several elastography techniques. Minimizing the maximum US pressure is essential in ophthalmology for safety reasons. For this purpose, we propose a pulse compression approach. It utilizes coded US emissions to generate shear waves where the energy is spread over a long emission, and then numerically compressed into a short, localized, and high-energy pulse. We used a 7.5-MHz single-element focused transducer driven by coded excitations where the amplitude is modulated by a linear frequency-swept square wave (1 to 7 kHz). An inverse filter approach was used for compression. We demonstrate the feasibility of performing shear wave elastography measurements in tissue-mimicking phantoms at low US pressures (mechanical index <0.6).

A composite set of ovarian tissue features extracted from photoacoustic spectral data, beam envelope, and co-registered ultrasound and photoacoustic images are used to characterize malignant and normal ovaries using logistic and support vector machine (SVM) classifiers. Normalized power spectra were calculated from the Fourier transform of the photoacoustic beamformed data, from which the spectral slopes and 0-MHz intercepts were extracted. Five features were extracted from the beam envelope and another 10 features were extracted from the photoacoustic images. These 17 features were ranked by their p-values from t-tests on which a filter type of feature selection method was used to determine the optimal feature number for final classification. A total of 169 samples from 19 ex vivo ovaries were randomly distributed into training and testing groups. Both classifiers achieved a minimum value of the mean misclassification error when the seven features with lowest p-values were selected. Using these seven features, the logistic and SVM classifiers obtained sensitivities of 96.39 ± 3.35% and 97.82 ± 2.26%, and specificities of 98.92 ± 1.39% and 100%, respectively, for the training group. For the testing group, logistic and SVM classifiers achieved sensitivities of 92.71 ± 3.55% and 92.64 ± 3.27%, and specificities of 87.52 ± 8.78% and 98.49 ± 2.05%, respectively.

The production of accurate and independent images of the changes in concentration of oxyhemoglobin and deoxyhemoglobin by diffuse optical imaging is heavily dependent on which wavelengths of near-infrared light are chosen to interrogate the target tissue. Although wavelengths can be selected by theoretical methods, in practice the accuracy of reconstructed images will be affected by wavelength-specific and system-specific factors such as laser source power and detector sensitivity. We describe the application of a data-driven approach to optimum wavelength selection for the second generation of University College London’s multichannel, time-domain optical tomography system (MONSTIR II). By performing a functional activation experiment using 12 different wavelengths between 690 and 870 nm, we were able to identify the combinations of 2, 3, and 4 wavelengths which most accurately reproduced the results obtained using all 12 wavelengths via an imaging approach. Our results show that the set of 2, 3, and 4 wavelengths which produce the most accurate images of functional activation are [770, 810], [770, 790, 850], and [730, 770, 810, 850] respectively, but also that the system is relatively robust to wavelength selection within certain limits. Although these results are specific to MONSTIR II, the approach we developed can be applied to other multispectral near-infrared spectroscopy and optical imaging systems.

Functional near-infrared spectroscopy (fNIRS) detects hemodynamic responses in the cerebral cortex by transcranial spectroscopy. However, measurements recorded by fNIRS not only consist of the desired hemodynamic response but also consist of a number of physiological noises. Because of these noises, accurately detecting the regions that have an activated hemodynamic response while performing a task is a challenge when analyzing functional activity by fNIRS. In order to better detect the activation, we designed a multiscale analysis based on wavelet coherence. In this method, the experimental paradigm was expressed as a binary signal obtained while either performing or not performing a task. We convolved the signal with the canonical hemodynamic response function to predict a possible response. The wavelet coherence was used to investigate the relationship between the response and the data obtained by fNIRS at each channel. Subsequently, the coherence within a region of interest in the time-frequency domain was summed to evaluate the activation level at each channel. Experiments on both simulated and experimental data demonstrated that the method was effective for detecting activated channels hidden in fNIRS data.

Counting morphologically normal cells in human red blood cells (RBCs) is extremely beneficial in the health care field. We propose a three-dimensional (3-D) classification method of automatically determining the morphologically normal RBCs in the phase image of multiple human RBCs that are obtained by off-axis digital holographic microscopy (DHM). The RBC holograms are first recorded by DHM, and then the phase images of multiple RBCs are reconstructed by a computational numerical algorithm. To design the classifier, the three typical RBC shapes, which are stomatocyte, discocyte, and echinocyte, are used for training and testing. Nonmain or abnormal RBC shapes different from the three normal shapes are defined as the fourth category. Ten features, including projected surface area, average phase value, mean corpuscular hemoglobin, perimeter, mean corpuscular hemoglobin surface density, circularity, mean phase of center part, sphericity coefficient, elongation, and pallor, are extracted from each RBC after segmenting the reconstructed phase images by using a watershed transform algorithm. Moreover, four additional properties, such as projected surface area, perimeter, average phase value, and elongation, are measured from the inner part of each cell, which can give significant information beyond the previous 10 features for the separation of the RBC groups; these are verified in the experiment by the statistical method of Hotelling’s T-square test. We also apply the principal component analysis algorithm to reduce the dimension number of variables and establish the Gaussian mixture densities using the projected data with the first eight principal components. Consequently, the Gaussian mixtures are used to design the discriminant functions based on Bayesian decision theory. To improve the performance of the Bayes classifier and the accuracy of estimation of its error rate, the leaving-one-out technique is applied. Experimental results show that the proposed method can yield good results for calculating the percentage of each typical normal RBC shape in a reconstructed phase image of multiple RBCs that will be favorable to the analysis of RBC-related diseases. In addition, we show that the discrimination performance for the counting of normal shapes of RBCs can be improved by using 3-D features of an RBC.

We reconstruct the three-dimensional shape and location of the retinal vascular network from commercial spectral-domain (SD) optical coherence tomography (OCT) data. The two-dimensional location of retinal vascular network on the eye fundus is obtained through support vector machines classification of properly defined fundus images from OCT data, taking advantage of the fact that on standard SD-OCT, the incident light beam is absorbed by hemoglobin, creating a shadow on the OCT signal below each perfused vessel. The depth-wise location of the vessel is obtained as the beginning of the shadow. The classification of crossovers and bifurcations within the vascular network is also addressed. We illustrate the feasibility of the method in terms of vessel caliber estimation and the accuracy of bifurcations and crossovers classification.

Raman spectroscopy is an optical method providing sample molecular composition, which can be analyzed (by point measurements) or spatially mapped by Raman imaging. These provide different information, signal-to-noise ratios, and require different acquisition times. Here, we quantitatively assess Raman spectral features and compare the two measurement methods by multivariate analysis. We also propose a hybrid method: scanning the beam through the sample but optically binning the signal at one location on the detector. This approach generates significantly more useful spectral signals in terms of peak visibility and statistical information. Additionally, by combination with a complementary imaging mode such as quantitative phase microscopy, hybrid imaging allows high throughput and robust spectral analysis while retaining sample spatial information. We demonstrate the improved ability to discriminate between cell lines when using hybrid scanning compared to typical point mode measurements, by quantitatively evaluating spectra taken from two macrophage-like cell lines. Hybrid scanning also provides better classification capability than the full Raman imaging mode, while providing higher signal-to-noise signals with shorter acquisition times. This hybrid imaging approach is suited for various applications including cytometry, cancer versus noncancer detection, and label-free discrimination of cell types or tissues.

A noninvasive high resolving power quantum microscope would facilitate progress in the areas of research and development in biosciences as well as in the area of biomedical technology. Longer-wavelength microscopes, i.e., visible or near-infrared, can provide noninvasive features. On the other hand, shorter wavelengths, i.e., in the ultraviolet, can provide better resolving power. We propose the development of both a noninvasive and high resolving power quantum microscope by using two-color entangled photon ghost imaging technology.

Functional brain imaging techniques require accurate co-registration to anatomical images to precisely identify the areas being activated. Many of them, including diffuse optical imaging, rely on scalp-placed recording sensors. Fiducial alignment is an effective and rapid method for co-registering scalp sensors onto anatomy, but is quite sensitive to placement errors. Surface Euclidean distance minimization using the Levenberq-Marquart algorithm (LMA) has been shown to be very accurate when based on good initial guesses, such as precise fiducial alignment, but its accuracy drops substantially with fiducial placement errors. Here we compared fiducial and LMA co-registration methods to a new procedure, the iterative closest point-to-plane (ICP2P) method, using simulated and real data. An advantage of ICP2P is that it eliminates the need to identify fiducials and is, therefore, entirely automatic. We show that, typically, ICP2P is as accurate as fiducial-based LMA, but is less sensitive to initial placement errors. However, ICP2P is more sensitive to spatially correlated noise in the description of the head surface. Hence, the best technique for co-registration depends on the type of data available to describe the scalp and the surface defined by the recording sensors. Under optimal conditions, co-registration error using surface-fitting procedures can be reduced to ∼3  mm.

We performed a study of the nonlinear optical properties of chemically purified chitin and insect cuticle using two-photon excited autofluorescence (TPEF) and second-harmonic generation (SHG) microscopy. Excitation spectrum, fluorescence time, polarization sensitivity, and bleaching speed were measured. We have found that the maximum autofluorescence signal requires an excitation wavelength below 850 nm. At longer wavelengths, we were able to penetrate more than 150-μm deep into the sample through the chitinous structures. The excitation power was kept below 10 mW (at the sample) in order to diminish bleaching. The SHG from the purified chitin was confirmed by spectral- and time-resolved measurements. Two cave-dwelling, depigmented, insect species were analyzed and three-dimensional images of the cuticular structures were obtained.

Measurement and imaging of depolarization by polarization-sensitive optical coherence tomography (PS-OCT) requires averaging of Stokes vector elements within two- or three-dimensional (3-D) evaluation windows to obtain the degree of polarization uniformity (DOPU). By use of a PS-OCT system with an integrated retinal tracker, we analyze optimum conditions for depolarization imaging, data processing, and segmentation of depolarizing tissue in the human retina. The trade-offs between figures of merit like DOPU imaging sensitivity, efficiency, and susceptibility are evaluated in terms of 3-D resolution. The results are used for a new, detailed interpretation of PS-OCT high-resolution images of the human retinal pigment epithelium and Bruch’s membrane.

We present a swept source optical coherence tomography (OCT) system at 1060 nm equipped with a wavefront sensor at 830 nm and a deformable mirror in a closed-loop adaptive optics (AO) system. Due to the AO correction, the confocal profile of the interface optics becomes narrower than the OCT axial range, restricting the part of the B-scan (cross section) with good contrast. By actuating on the deformable mirror, the depth of the focus is changed and the system is used to demonstrate Gabor filtering in order to produce B-scan OCT images with enhanced sensitivity throughout the axial range from a Drosophila larvae. The focus adjustment is achieved by manipulating the curvature of the deformable mirror between two user-defined limits. Particularities of controlling the focus for Gabor filtering using the deformable mirror are presented.

We demonstrate the first application of the recently proposed method of optical palpation to in vivo imaging of human skin. Optical palpation is a tactile imaging technique that probes the spatial variation of a sample’s mechanical properties by producing an en face map of stress measured at the sample surface. This map is determined from the thickness of a translucent, compliant stress sensor placed between a loading element and the sample and is measured using optical coherence tomography. We assess the performance of optical palpation using a handheld imaging probe on skin-mimicking phantoms, and demonstrate its use on human skin lesions. Our results demonstrate the capacity of optical palpation to delineate the boundaries of lesions and to map the mechanical contrast between lesions and the surrounding normal skin.

An imaging probe is designed and developed by integrating a miniaturized charge-coupled diode camera and light-emitting diode light source, which enables evaluation of the iridocorneal region inside the eye. The efficiency of the prototype probe instrument is illustrated initially by using not only eye models, but also samples such as pig eye. The proposed methodology and developed scheme are expected to find potential application in iridocorneal angle documentation, glaucoma diagnosis, and follow-up management procedures.

Uveitis models in rodents are important in the investigation of pathogenesis in human uveitis and the development of appropriate therapeutic strategies for treatment. Quantitative monitoring of ocular inflammation in small animal models provides an objective metric to assess uveitis progression and/or therapeutic effects. We present a new application of optical coherence tomography (OCT) and OCT-based microangiography (OMAG) to a rat model of acute anterior uveitis induced by intravitreal injection of a killed mycobacterial extract. OCT/OMAG is used to provide noninvasive three-dimensional imaging of the anterior segment of the eyes prior to injection (baseline) and two days post-injection (peak inflammation) in rats with and without steroid treatments. OCT imaging identifies characteristic structural and vascular changes in the anterior segment of the inflamed animals when compared to baseline images. Characteristics of inflammation identified include anterior chamber cells, corneal edema, pupillary membranes, and iris vasodilation. In contrast, no significant difference from the control is observed for the steroid-treated eye. These findings are compared with the histology assessment of the same eyes. In addition, quantitative measurements of central corneal thickness and iris vessel diameter are determined. This pilot study demonstrates that OCT-based microangiography promises to be a useful tool for the assessment and management of uveitis in vivo.

Quantitative assessment of wound tissue ischemia, perfusion, and inflammation provides critical information for appropriate detection, staging, and treatment of chronic wounds. However, few methods are available for simultaneous assessment of these tissue parameters in a noninvasive and quantitative fashion. We integrated hyperspectral, laser speckle, and thermographic imaging modalities in a single-experimental setup for multimodal assessment of tissue oxygenation, perfusion, and inflammation characteristics. Algorithms were developed for appropriate coregistration between wound images acquired by different imaging modalities at different times. The multimodal wound imaging system was validated in an occlusion experiment, where oxygenation and perfusion maps of a healthy subject’s upper extremity were continuously monitored during a postocclusive reactive hyperemia procedure and compared with standard measurements. The system was also tested in a clinical trial where a wound of three millimeters in diameter was introduced on a healthy subject’s lower extremity and the healing process was continuously monitored. Our in vivo experiments demonstrated the clinical feasibility of multimodal cutaneous wound imaging.

Hyperspectral imaging (HSI) is a new technology that obtains spectroscopic information and renders it in image form. This study examined the difference in the spectral reflectance (SR) of gastric tumors and normal mucosa recorded with a hyperspectral camera equipped with HSI technology and attempted to determine the specific wavelength that is useful for the diagnosis of gastric cancer. A total of 104 gastric tumors removed by endoscopic submucosal dissection from 96 patients at Yamaguchi University Hospital were recorded using a hyperspectral camera. We determined the optimal wavelength and the cut-off value for differentiating tumors from normal mucosa to establish a diagnostic algorithm. We also attempted to highlight tumors by image processing using the hyperspectral camera’s analysis software. A wavelength of 770 nm and a cut-off value of 1/4 the corrected SR were selected as the respective optimal wavelength and cut-off values. The rates of sensitivity, specificity, and accuracy of the algorithm’s diagnostic capability were 71%, 98%, and 85%, respectively. It was possible to enhance tumors by image processing at the 770-nm wavelength. HSI can be used to measure the SR in gastric tumors and to differentiate between tumorous and normal mucosa.

Near infrared (NIR) fluorescence imaging has shown great potential for various clinical procedures, including intraoperative image guidance. However, existing NIR fluorescence imaging systems either have a large footprint or are handheld, which limits their usage in intraoperative applications. We present a compact NIR fluorescence imaging system (NFIS) with an image overlay solution based on threshold detection, which can be easily integrated with a goggle display system for intraoperative guidance. The proposed NFIS achieves compactness, light weight, hands-free operation, high-precision superimposition, and a real-time frame rate. In addition, the miniature and ultra-lightweight light-emitting diode tracking pod is easy to incorporate with NIR fluorescence imaging. Based on experimental evaluation, the proposed NFIS solution has a lower detection limit of 25 nM of indocyanine green at 27 fps and realizes a highly precise image overlay of NIR and visible images of mice in vivo. The overlay error is limited within a 2-mm scale at a 65-cm working distance, which is highly reliable for clinical study and surgical use.

Using internal illumination with an optical fiber in the oral cavity, we demonstrate, for the first time, photoacoustic computed tomography (PACT) of the deep brain of rats in vivo. The experiment was performed on a full-ring-array PACT system, with the capability of providing high-speed cross-sectional imaging of the brain. Compared with external illumination through the cranial skull, internal illumination delivers more light to the base of the brain. Consequently, in vivo photoacoustic images clearly reveal deep brain structures such as the hypothalamus, brain stem, and cerebral medulla.

The liver is a major organ responsible for performing xenobiotic metabolism. In this process, xenobiotic is uptaken and processed in hepatocytes and subsequently excreted into the bile canaliculi. However, the intracellular heterogeneity in such metabolic processes is not known. We use the molecular probe 6-carboxyfluorescein diacetate (6-CFDA) to investigate xenobiotic metabolism in hepatocytes with intravital multiphoton fluorescence microscopy. 6-CFDA is processed by intracellular esterase to fluorescent 6-CF, which can be imaged and quantified. We found that compared to the nucleus, cytoplasmic 6-CF fluorescence intensity reached a maximum earlier (cytoplasm: 11.3±4.4  min; nucleus: 14.7±4.9  min) following 6-CFDA injection. We also found a slight difference in the rate of 6-CFDA metabolism as the rates of 6-CF decay at rates of 1.43±0.75 and 1.27±0.72  photons/min for the cytoplasm and nucleus, respectively. These results indicate that molecular transport to the nucleus is additionally hindered and can affect drug transport there.

Quantitative methods for noninvasive diagnosis of scars are a challenging issue in medicine. This work aims to implement a texture analysis method for quantitatively discriminating abnormal scars from normal scars based on second-harmonic generation (SHG) images. A local difference local binary pattern (LD-LBP) operator combined with a wavelet transform was explored to extract diagnosis features from scar SHG images that were related to the alteration in collagen morphology. Based on the quantitative parameters including the homogeneity, directional and coarse features in SHG images, the scar collagen SHG images were classified into normal or abnormal scars by a support vector machine classifier in a leave-one-out cross-validation procedure. Our experiments and data analyses demonstrated apparent differences between normal and abnormal scars in terms of their morphological structure of collagen. By comparing with gray level co-occurrence matrix, wavelet transform, and combined basic local binary pattern and wavelet transform with respect to the accuracy and receiver operating characteristic analysis, the method proposed herein was demonstrated to achieve higher accuracy and more reliable classification of SHG images. This result indicated that the extracted texture features with the proposed method were effective in the classification of scars. It could provide assistance for physicians in the diagnostic process.

Vascular leakage is an important feature of various disease conditions. In vivo optical imaging provides a great opportunity for the evaluation of this phenomenon. In the present study, we focus on the development and validation of a near-infrared (NIR) imaging formula to allow reliable, cost-efficient evaluation of vascular leakage in diverse species using the existing small-animal fluorescence imaging technology. IR-676, a moderately hydrophobic NIR cyanine dye, was doped into self-assembling aqueous micelles using a widely employed and safe nonionic emulsifier (Kolliphor HS 15), and was tested in several acute and chronic inflammatory disease models in both mice and rats. The imaging formula is stable and exerts no acute toxic effects in vitro. It accumulated specifically in the inflamed regions in all models, which could be demonstrated by both conventional epifluorescence imaging, and fluorescence tomography both as a standalone technique and also by merging it with computed tomography scans. Ex vivo verification of dye accumulation by confocal fluorescence microscopy was also possible. The present formula allows sensitive and specific detection of inflammatory plasma leakage in diverse models. Its potential for imaging larger animals was also demonstrated. IR-676-doped micelles offer an excellent opportunity to image inflammatory vascular leakage in various models and species.

Pulsed photothermal radiometry (PPTR) allows noninvasive determination of laser-induced temperature depth profiles in optically scattering layered structures. The obtained profiles provide information on spatial distribution of selected chromophores such as melanin and hemoglobin in human skin. We apply the described approach to study time evolution of incidental bruises (hematomas) in human subjects. By combining numerical simulations of laser energy deposition in bruised skin with objective fitting of the predicted and measured PPTR signals, we can quantitatively characterize the key processes involved in bruise evolution (i.e., hemoglobin mass diffusion and biochemical decomposition). Simultaneous analysis of PPTR signals obtained at various times post injury provides an insight into the variations of these parameters during the bruise healing process. The presented methodology and results advance our understanding of the bruise evolution and represent an important step toward development of an objective technique for age determination of traumatic bruises in forensic medicine.

We present the prediction of malaria infection in human plasma using Raman spectroscopy. Raman spectra of malaria-infected samples are compared with those of healthy and dengue virus infected ones for disease recognition. Raman spectra were acquired using a laser at 532 nm as an excitation source and 10 distinct spectral signatures that statistically differentiated malaria from healthy and dengue-infected cases were found. A multivariate regression model has been developed that utilized Raman spectra of 20 malaria-infected, 10 non-malarial with fever, 10 healthy, and 6 dengue-infected samples to optically predict the malaria infection. The model yields the correlation coefficient r² value of 0.981 between the predicted values and clinically known results of trainee samples, and the root mean square error in cross validation was found to be 0.09; both these parameters validated the model. The model was further blindly tested for 30 unknown suspected samples and found to be 86% accurate compared with the clinical results, with the inaccuracy due to three samples which were predicted in the gray region. Standard deviation and root mean square error in prediction for unknown samples were found to be 0.150 and 0.149, which are accepted for the clinical validation of the model.

Fluorescence molecular tomography (FMT) imaging can be used to determine the location, size, and biodistribution of fluorophore biomarkers inside tissues. Yet when using FMT in the reflectance geometry it is challenging to accurately localize fluorophores. A depth perturbation method is proposed to determine the centroid of fluorophore inside a tissue-like medium. Through superposition of a known thin optical phantom onto the medium surface, the fluorophore depth is deliberately perturbed and signal localization is improved in a stable way. We hypothesize that the fluorophore centroid can be better localized through use of this fluorescent intensity variation resulting from the depth perturbation. This hypothesis was tested in tissue-like phantoms. The results show that a small-size fluorophore inclusion (1.2  mm³ volume, depth up to 4.8 mm) can be localized by the method with an error of 0.2 to 0.3 mm. The method is also proven to be capable of handling multiple fluorescent inclusion conditions with the assistance of other strategies. Additionally, our further studies showed that the method’s performance in the presence of background fluorophores indicated that the small inclusion could be located at a 1.8 (3.8) mm depth with accurate localization only when its concentration was not <10 (100) times the background level.

Reduced nicotinamide adenine dinucleotide (NADH) fluorescence lifetime has been broadly used as a metabolic indicator for stem cell imaging. However, the direct relationship between NADH fluorescence lifetime and metabolic pathway and activity remains to be clarified. In this study, we measured the NADH fluorescence lifetime of human mesenchymal stem cells (hMSCs) as well as the metabolic indictors, such as adenosine triphosphate (ATP) level, oxygen consumption, and lactate release, up to 4 weeks under normal osteogenic differentiation and oxidative phosphorylation-attenuated/inhibited differentiation by oligomycin A (OA) treatment. NADH fluorescence lifetime was positively correlated with oxygen consumption and ATP level during energy transformation from glycolysis to oxidative phosphorylation. Under OA treatment, oxidative phosphorylation was attenuated/inhibited (i.e., oxygen consumption remained the same as controls or lower), cells showed attenuated differentiation under glycolysis, and NADH fluorescence lifetime change was not detected. Increased expression of the overall complex proteins was observed in addition to Complex I. We suggested special caution needs to be exercised while interpreting NADH fluorescence lifetime signal in terms of stem cell differentiation.

This work reports that the ablation volume and rate of porcine skin changed significantly with the change of skin water content. Under the same laser irradiation conditions (532 nm Nd:YAG laser, pulse width = 11.5  ns, pulse energy = 1.54  J, beam radius = 0.54  mm), the ablation volume dropped by a factor of 4 as the skin water content decreased from 40 wt. % (native) to 19 wt. % with a change in the ablation rate below and above around 25 wt. %. Based on the ablation characteristics observed by in situ shadowgraph images and the calculated tissue temperatures, it is considered that an explosive rupture by rapid volumetric vaporization of water is responsible for the ablation of the high water content of skin, whereas thermal disintegration of directly irradiated surface layer is responsible for the low water content of skin.

The present study evaluated the intrapulpal thermal changes that occurred during the treatment of the root surfaces with a laser system emitting Er,Cr:YSGG 2780- and 940-nm diode laser irradiation in an alternating sequence. Thirty single-rooted human teeth were collected. The teeth were divided into three groups (n=10 each) and irradiated with Er,Cr:YSGG alone or combined with a 940-nm diode laser. To investigate the intrapulpal temperature changes, specimens were embedded in a resin block with a set of thermocouples introduced at different positions within the root canals. The first group was irradiated with only Er,Cr:YSGG (25 mJ, 50 Hz, 50  μs pulse duration, water and air spray); the second group was irradiated with Er,Cr:YSGG (same setting) and a 940-nm diode (2 W, chopped mode with 20% duty cycle); the third group was irradiated with Er,Cr:YSGG (same setting) and a diode (2 W, chopped mode with 50% duty cycle). During all irradiations, thermal changes were recorded in real time with thermocouples. While group 3 showed thermal rises on average of 1.68±0.98°C in the pulp chamber, groups 1 and 2 showed average temperature rises of <0.5°C. The combined laser emission of 2780 and 940 nm is a promising way for root surface debridement without inducing intrapulpal thermal damage when using an appropriate water/air spray. All measured temperatures were considerably below the critical value of 5.6°C.

This PDF file contains the editorial “Super-Resolution Microscopy Techniques in the Neurosciences” for JBO Vol. 20 Issue 01