Optical clearing agents (OCAs) and many chemicals are widely used in functional diagnosis of skin tissues. Numerous studies are associated with the transcutaneous diffusion of OCA in epidermal, dermal, and hypodermal tissues, which results in changing their optical properties. In addition, an objective approach that is suitable for screening the influence of utilized OCA, as well as various chemical agents, synthetics, and nanomaterials, on blood and lymph flows is highly desirable. In our study, a highly sensitive laser speckle imaging (LSI) system and fluorescent intravital microscopy (FIM) were used team-wise to inspect the acute skin vascular permeability reaction in mouse ear during the local application of OCA on the skin surface. Fluorescent contrast material administrated intravenously was used for quantitatively assessing the intensity of vascular permeability reaction and the strength of skin irritation. The obtained results suggest that a combined use of LSI and FIM is highly effective for monitoring the cutaneous vascular permeability reaction, with great potential for assessment of allergic reactions of skin in response to interactions with chemical substances.

Early stage porcine parthenogenetic embryos were evaluated for metabolic activity using a biodynamic microscope (BDM) that images dynamic light scattering using low-coherence digital holography. The microscope has a 45-deg illumination configuration that reduces specular background for the imaging of small translucent samples. The off-axis illumination is compatible with coherence-gated imaging because of volumetric light scattering in which the coherence plane is tilted at half the illumination angle in a three-dimensional tissue target. The BDM was used to profile the viability of porcine parthenotes with normal and with inhibited mitochondrial adenosine triphosphate (ATP) production using Doppler fluctuation spectroscopy. The ATP concentrations in the parthenotes, which are indicative of developmental potential, were validated by a conventional bioluminescence assay. Biodynamic classifications achieved ∼80  %   accuracy correlating sample ATP treatment, providing a quick, label-free surrogate measurement to replace invasive metabolic assays as a candidate for evaluating quality of early embryos in the assisted reproductive technology setting.

Affluent blood flow through a complicated net of vessels supplies skeletal bone tissue with oxygen and nutrients. Due to accidental events or physiological processes, the blood supply might be deficient or even disrupted, and the healthy bone decays in a process that, for the hip location, is denoted as osteonecrosis of the femoral head (ONFH) or avascular femoral head necrosis. Early diagnosis is important for the prognosis. X-ray-based imaging, such as CT or MRI, is not of much value for the early detection. As the decay theoretically is associated with the development of gas-filled pores, gas analysis should have diagnostic value. We have introduced gas in scattering media absorption spectroscopy, as a complementary modality. Eighteen extracted femoral joint heads, diseased as well as normal, were investigated. Diseased samples are associated with clear signals due to water vapor, whereas the normal ones largely lack such features. The results suggest that free water vapor could serve as an early indicator of pore development and thus as a promising predictor of ONFH pathological changes, once the technique has been fully refined.

Computational models predicting cell damage responses to transient temperature rises generated by exposure to lasers have implemented the damage integral (Ω), which time integrates the chemical reaction rate constant described by Arrhenius. However, few published reports of empirical temperature histories (thermal profiles) correlated with damage outcomes at the cellular level are available to validate the breadth of applicability of the damage integral. In our study, an analysis of photothermal damage rate processes in cultured retinal pigment epithelium cells indicated good agreement between temperature rise, exposure duration (τ), and threshold cellular damage. Full-frame thermograms recorded at high magnification during laser exposures were overlaid with fluorescence damage images taken 1 h postexposure. From the image overlays, pixels of the thermogram correlated with the boundary of cell death were used to extract threshold thermal profiles. Assessing photothermal responses at these boundaries standardized all data points, irrespective of laser irradiance, damage size, or optical and thermal properties of the cells. These results support the hypothesis that data from boundaries of cell death were equivalent to a minimum visible lesion, where the damage integral approached unity (Ω  =  1) at the end of the exposure duration. Empirically resolved Arrhenius coefficients for use in the damage integral determined from exposures at wavelengths of 2  μm and 532 nm and durations of 0.05–20 s were consistent with literature values. Varying ambient temperature (T_amb) between 20°C and 40°C during laser exposure did not change the τ-dependent threshold peak temperature (T_p). We also show that, although threshold laser irradiance varied due to pigmentation differences, threshold temperatures were irradiance independent.

Plasmonic nanoparticles (PNPs) continue to see increasing use in biophotonics for a variety of applications, including cancer detection and treatment. Several PNP-based approaches involve the generation of highly transient nanobubbles due to pulsed laser-induced vaporization and cavitation. While much effort has been devoted to elucidating the mechanisms behind bubble generation with spherical gold nano particles, the effects of particle shape on bubble generation thresholds are not well understood, especially in the nanosecond pulse regime. Our study aims to compare the bubble generation thresholds of gold nanospheres, gold nanorods, and silica-core gold nanoshells with different sizes, resonances, and surface coatings. Bubble generation is detected using a multimodality microscopy platform for simultaneous, nanosecond resolution pump-probe imaging, integrated scattering response, and acoustic transient detection. Nanoshells and large (40-nm width) nanorods were found to have the lowest thresholds for bubble generation, and in some cases they generated bubbles at radiant exposures below standard laser safety limits for skin exposure. This has important implications for both safety and performance of techniques employing pulsed lasers and PNPs.

Quantitative photoacoustic tomography aims to recover maps of the local concentrations of tissue chromophores from multispectral images. While model-based inversion schemes are promising approaches, major challenges to their practical implementation include the unknown fluence distribution and the scale of the inverse problem. We describe an inversion scheme based on a radiance Monte Carlo model and an adjoint-assisted gradient optimization that incorporates fluence-dependent step sizes and adaptive moment estimation. The inversion is shown to recover absolute chromophore concentrations, blood oxygen saturation, and the Grüneisen parameter from in silico three-dimensional phantom images for different radiance approximations. The scattering coefficient is assumed to be homogeneous and known a priori.

Hyperspectral imaging has shown great potential for optical skin analysis by providing noninvasive, pixel-by-pixel surface measurements from which, applying an optical model, information such as melanin concentration and total blood volume fraction can be mapped. Such applications have been successfully performed on small flat skin areas, but existing methods are not suited to large areas such as an organ or a face, due to the difficulty of ensuring homogeneous illumination on complex three-dimensional (3-D) objects, which leads to errors in the maps. We investigate two methods to account for these irradiance variations on a face. The first one relies on a radiometric correction of the irradiance, using 3-D information on the face’s shape acquired by combining the hyperspectral camera with a 3-D scanner; the second relies on an optimization metric used in the map computation, which is invariant to irradiance. We discuss the advantages and drawbacks of the two methods, after having presented in detail the whole acquisition setup, which has been designed to provide high-resolution images with a short acquisition time, as required for live surface measurements of complex 3-D objects such as the face.

Lipid composition of atherosclerotic plaques is considered to be highly related to plaque vulnerability. Therefore, a specific diagnostic or imaging modality that can sensitively evaluate plaques’ necrotic core is desirable in atherosclerosis imaging. In this regard, intravascular photoacoustic (IVPA) imaging is an emerging plaque detection technique that provides lipid-specific chemical information from an arterial wall with great optical contrast and long acoustic penetration depth. While, in the near-infrared window, a 1210-nm optical source is usually chosen for IVPA applications since lipids exhibit a strong absorption peak at that wavelength, the sensitivity problem arises in the conventional single-ended systems as other arterial tissues also show some degree of absorption near that spectral region, thereby generating undesirably interfering photoacoustic (PA) signals. A theory of the high-frequency frequency-domain differential photoacoustic radar (DPAR) modality is introduced as a unique detection technique for accurate and molecularly specific evaluation of vulnerable plaques. By assuming two low-power continuous-wave optical sources at ∼1210 and ∼970  nm in a differential manner, DPAR theory and the corresponding simulation/experiment studies suggest an imaging modality that is only sensitive and specific to the spectroscopically defined imaging target, cholesterol.

The fluorescent imaging agent IS-001 was determined to be well tolerated in all subjects and has the potential to provide ureter visualization throughout minimally invasive hysterectomy procedures. This study was conducted to evaluate clinical safety and efficacy of a real-time ureter visualization technique for use during hysterectomy surgery. The study drug appears safe, is renally excreted, and allows enhanced ureter visualization when imaged with a clinically approved near-infrared sensitive endoscope. This is a first-in-human study showing preliminary results that the drug is safe and effective during surgery for improved ureter visualization.

A clinical need exists for low-cost and noninvasive imaging tools capable of detecting inflammation in the joints of inflammatory arthritis patients. Previous studies have reported an optical contrast between inflamed and noninflamed joints resulting from distinct absorption and scattering properties. Accurate classification using nonocclusion-based continuous wave, transillumination imaging was limited to patient-specific changes during follow-up examination as opposed to single time-point examination, which was attributed to high intersubject variability. In distinction from previous work, optical images were acquired from the dorsal side with illumination on the palmar side and features about the spatial distribution of transmitted light along the joint were assessed using a normalized Fourier transform method. Results using this approach demonstrated an area under receiver operator curve of up to 0.888 for detecting inflammation in a pilot study involving single time-point examination of 144 joints from 21 rheumatology patients. This workflow may enable future development of clinically viable, low-cost devices for assessing inflammation in arthritis patients, without the need for cuff occlusion or comparison to baseline.

Although the luminescence of water at a lower energy than the Cerenkov-light (CL) threshold has been found for various types of radiation, the fractions of the luminescence of water to the total produced light have not been obvious for radiations at a higher energy than the CL threshold because it is difficult to separate these two types of light. Thus, we used a Monte Carlo simulation to estimate the fractions of the luminescence of water for various types of radiation at a higher energy than the CL threshold to confirm the major component of the produced light. After we confirmed that the estimated light production of the luminescence of water could adequately simulate the experimental results, we calculated the produced light photons of this luminescence and the CL from water for protons (170 MeV), carbon ions (330 MeV/n), high-energy x-ray (6 MV) from a linear accelerator (LINAC), high-energy electrons (9 MeV) from LINAC, positrons (F-18, C-11, O-15, and N-13), and high-energy gamma photon radionuclides (Co-60). For protons, the major fraction of the produced light was the luminescence of water in addition to the CL from the prompt gamma photons produced by the nuclear interactions. For carbon ions, the major fraction of the produced light was the luminescence of water and the CL produced by the secondary electrons in addition to the prompt gamma photons produced by the nuclear interactions. For high-energy x-ray and electrons from LINAC, the fractions of luminescence of water were ∼0.1  %   to 0.2%. The fractions of luminescence of water for positrons were 0.2% to 1.5% and that for Co-60 was 0.4%. We conclude that the major fractions of light produced from x-ray and electrons from LINAC, positron radionuclides, and the Co-60 source are CL, with fractions of the luminescence of water from <0.1  %   to 1.5%.

We report a compact rigid instrument capable of delivering en-face optical coherence tomography (OCT) images alongside (epi)-fluorescence endomicroscopy (FEM) images by means of a robotic scanning device. Two working imaging channels are included: one for a one-dimensional scanning, forward-viewing OCT probe and another for a fiber bundle used for the FEM system. The robotic scanning system provides the second axis of scanning for the OCT channel while allowing the field of view (FoV) of the FEM channel to be increased by mosaicking. The OCT channel has resolutions of 25  /  60  μm (axial/lateral) and can provide en-face images with an FoV of 1.6  ×  2.7  mm². The FEM channel has a lateral resolution of better than 8  μm and can generate an FoV of 0.53  ×  3.25  mm² through mosaicking. The reproducibility of the scanning was determined using phantoms to be better than the lateral resolution of the OCT channel. Combined OCT and FEM imaging were validated with ex-vivo ovine and porcine tissues, with the instrument mounted on an arm to ensure constant contact of the probe with the tissue. The OCT imaging system alone was validated for in-vivo human dermal imaging with the handheld instrument. In both cases, the instrument was capable of resolving fine features such as the sweat glands in human dermal tissue and the alveoli in porcine lung tissue.

A membrane potential change in cells is accompanied with mechanical deformation. This electromechanical response can play a significant role in regulating action potential in neurons and in controlling voltage-gated ion channels. However, measuring this subtle deformation in mammalian cells has been a difficult task. We show a plasmonic imaging method to image mechanical deformation in single cells upon a change in the membrane potential. Using this method, we have studied the electromechanical response in mammalian cells and have observed the local deformation within the cells that are associated with cell–substrate interactions. By analyzing frequency dependence of the response, we have further examined the electromechanical deformation in terms of mechanical properties of cytoplasm and cytoskeleton. We demonstrate a plasmonic imaging approach to quantify the electromechanical responses of single mammalian cells and determine local variability related to cell–substrate interactions.

Noninvasive and real-time visualization of the thoracoepigastric veins (TVs) of living mice was demonstrated by using two-photon excitation (TPE) optical imaging with a Eu-luminescent polymeric nanoagent as the angiographic contrast. The spatiotemporal evolution of the polymeric nanoagent in TVs was monitored for up to 2 h by TPE time-resolved (TPE-TR) bioimaging, which is free from the interference of tissue autofluorescence. A wide field-of-view covering the thoracoabdominal region allowed the visualization of the entire TV network with an imaging depth of 1 to 2 mm and a lateral resolution of 80 μm at submillimeter. Detailed analysis of the uptake, transport, and clearance processes of the polymeric nanoagent revealed a clearance time constant of ∼30  min and an apparent clearance efficiency of 80% to 90% for the nanoagent in both axial and lateral TVs. TPE-TR imaging of the dissected internal organs proved that the liver is mainly responsible for the sequestration of the nanoagent, which is consistent with the apparent retention efficiency of liver, ∼32  %  , as determined by the real-time in vivo TV imaging. We demonstrate the potency of TPE-TR modality in the pharmacokinetics imaging of the peripheral vascular systems of animal models, which can be beneficial for related nanotheranostics study.

We present a multimodal visible light optical coherence microscopy (OCM) and fluorescence imaging (FI) setup. Specification and phantom measurements were performed to characterize the system. Two applications in neuroimaging were investigated. First, curcumin-stained brain slices of a mouse model of Alzheimer’s disease were examined. Amyloid-beta plaques were identified based on the fluorescence of curcumin, and coregistered morphological images of the brain tissue were provided by the OCM channel. Second, human brain tumor biopsies retrieved intraoperatively were imaged prior to conventional neuropathologic work-up. OCM revealed the three-dimensional structure of the brain parenchyma, and FI added the tumor tissue-specific contrast. Attenuation coefficients computed from the OCM data and the florescence intensity values were analyzed and showed a statistically significant difference for 5-aminolevulinic acid (5-ALA)-positive and -negative brain tissues. OCM findings correlated well with malignant hot spots within brain tumor biopsies upon histopathology. The combination of OCM and FI seems to be a promising optical imaging modality providing complementary contrast for applications in the field of neuroimaging.

Optical coherence tomography (OCT) is a powerful tool in ophthalmology that provides in vivo morphology of the retinal layers and their light scattering properties. The directional (angular) reflectivity of the retinal layers was investigated with focus on the scattering from retinal pigment epithelium (RPE). The directional scattering of the RPE was studied in three mice strains with three distinct melanin concentrations: albino (BALB/c), agouti (129S1/SvlmJ), and strongly pigmented (C57BL/6J). The backscattering signal strength was measured with a directional OCT system in which the pupil entry position of the narrow OCT beam can be varied across the dilated pupil of the eyes of the mice. The directional reflectivity of other retinal melanin-free layers, including the internal and external limiting membranes, and Bruch’s membrane (albinos) were also measured and compared between the strains. The intensity of light backscattered from these layers was found highly sensitive to the angle of illumination, whereas the inner/outer segment (IS/OS) junctions showed a reduced sensitivity. The reflections from the RPE are largely insensitive in highly pigmented mice. The differences in directional scattering between strains shows that directionality decreases with an increase in melanin concentrations in RPE, suggesting increasing contribution of Mie scattering by melanosomes.

Real-time intraoperative image-guided cancer surgery promises to improve oncologic outcomes. Tumor-specific antibodies conjugated with near-infrared (NIR) fluorophores have demonstrated the potential to enhance visualization of solid tumor margins and metastatic disease; however, multiple challenges remain, including improvement in probe development for clinical utility. We have developed an NIR-IR800 dye on a PEGylated linker (sidewinder) conjugated to the humanized anti-carcinoembryonic antigen (CEA) antibody (M5A) with extended in vivo serum and tumor persistence. The anti-CEA M5A-sidewinder has a high dye-to-antibody ratio (average of 7 per antibody) that allows, in an orthotopic implanted human pancreatic cancer mouse model increased tumor fluorescence, higher tumor-to-background ratio and extends the surgical scheduling window compared to current antibody dye conjugates. These preclinical results demonstrate the potential of this probe for fluorescence-guided surgery of CEA-positive gastrointestinal cancers.

Collagen remodeling occurs in many prostate pathologies; however, the underlying structural architecture in both normal and diseased prostatic tissues is largely unexplored. Here, we use second-harmonic generation (SHG) microscopy to specifically probe the role of the proteoglycan decorin (Dcn) on collagen assembly in a wild type (wt) and Dcn null mouse (Dcn^{−    /    −}). Dcn is required for proper organization of collagen fibrils as it regulates size by forming an arch-like structure at the end of the fibril. We have utilized SHG metrics based on emission directionality (forward–backward ratio) and relative conversion efficiency, which are both related to the SHG coherence length, and found more disordered fibril organization in the Dcn^{−    /    −}. We have also used image analysis readouts based on entropy, multifractal dimension, and wavelet transforms to compare the collagen fibril/fiber architecture in the two models, where all these showed that the Dcn^{−    /    −} prostate comprised smaller and more disorganized collagen structures. All these SHG metrics are consistent with decreased SHG phase matching in the Dcn^{−    /    −} and are further consistent with ultrastructural analysis of collagen in this model in other tissues, which show a more random distribution of fibril sizes and their packing into fibers. As Dcn is a known tumor suppressor, this work forms the basis for future studies of collagen remodeling in both malignant and benign prostate disease.

Free tissue transfer (FTT) surgery for breast reconstruction following mastectomy has become a routine operation with high success rates. Although failure is low, it can have a devastating impact on patient recovery, prognosis, and psychological well-being. Continuous and objective monitoring of tissue oxygen saturation (StO₂) has been shown to reduce failure rates through rapid detection time of postoperative vascular complications. We have developed a pervasive wearable wireless device that employs near-infrared spectroscopy (NIRS) to continuously monitor FTT via StO₂ measurement. Previously tested on different models, the results of a clinical study are introduced. Our goal for the study is to demonstrate that the developed device can reliably detect StO₂ variations in a clinical setting: 14 patients were recruited. Advanced data analysis was performed on the StO₂ variations, the relative StO₂ gradient change, and the classification of the StO₂ within different clusters of blood occlusion level (from 0% to 100% at 25% step) based on previous studies made on a vascular phantom and animals. The outcomes of the clinical study concur with previous experimental results and the expected biological responses. This suggests that the device is able to correctly detect perfusion changes and provide real-time assessment on the viability of the FTT in a clinical setting.

This erratum corrects an error in “Modeling subdiffusive light scattering by incorporating the tissue phase function and detector numerical aperture.”