Efficient phenotyping of developmental defects in model organisms is critical for understanding the genetic specification of normal development and congenital abnormalities in humans. We previously reported that optical coherence tomography (OCT) combined with live embryo culture is a valuable tool for mouse embryo imaging and four-dimensional (4-D) cardiodynamic analysis; however, its capability for analysis of mouse mutants with cardiac phenotypes has not been previously explored. Here, we report 4-D (three-dimensional+time) OCT imaging and analysis of the embryonic heart in a Wdr19 mouse mutant, revealing a heart looping defect. Quantitative analysis of cardiac looping revealed a statistically significant difference between mutant and control embryos. Our results indicate that live 4-D OCT imaging provides a powerful phenotyping approach to characterize embryonic cardiac function in mouse models.

For fluorescence tomographic imaging of small animals, the liver is usually regarded as a low-scattering tissue and is surrounded by adipose, kidneys, and heart, all of which have a high scattering property. This leads to a breakdown of the diffusion equation (DE)–based reconstruction method as well as a heavy computational burden for the simplified spherical harmonics equation (SPN). Coupling the SPN and DE provides a perfect balance between the imaging accuracy and computational burden. The coupled third-order SPN and DE (CSDE)-based reconstruction method is developed for fluorescence tomographic imaging. This is achieved by doubly using the CSDE for the excitation and emission processes of the fluorescence propagation. At the same time, the finite-element method and hybrid multilevel regularization strategy are incorporated in inverse reconstruction. The CSDE-based reconstruction method is first demonstrated with a digital mouse-based liver cancer simulation, which reveals superior performance compared with the SPN and DE-based methods. It is more accurate than the DE-based method and has lesser computational burden than the SPN-based method. The feasibility of the proposed approach in applications of in vivo studies is also illustrated with a liver cancer mouse-based in situ experiment, revealing its potential application in whole-body imaging of small animals.

RGB (red-green-blue) technique for mapping skin chromophores by smartphones is proposed and studied. Three smartphones of different manufacturers were tested on skin phantoms and in vivo on benign skin lesions using a specially designed light source for illumination. Hemoglobin and melanin indices obtained by these smartphones showed differences in both tests. In vitro tests showed an increment of hemoglobin and melanin indices with the concentration of chromophores in phantoms. In vivo tests indicated higher hemoglobin index in hemangiomas than in nevi and healthy skin, and nevi showed higher melanin index compared to the healthy skin. Smartphones that allow switching off the automatic camera settings provided useful data, while those with “embedded” automatic settings appear to be useless for distant skin chromophore mapping.

Intrinsic optical signal (IOS) imaging promises to be a noninvasive method for high-resolution examination of retinal physiology, which can advance the study and diagnosis of eye diseases. While specialized optical instruments are desirable for functional IOS imaging of retinal physiology, in depth understanding of multiple IOS sources in the complex retinal neural network is essential for optimizing instrument designs. We provide a brief overview of IOS studies and relationships in rod outer segment suspensions, isolated retinas, and intact eyes. Recent developments of line-scan confocal and functional optical coherence tomography (OCT) instruments have allowed in vivo IOS mapping of photoreceptor physiology. Further improvements of the line-scan confocal and functional OCT systems may provide a feasible solution to pursue functional IOS mapping of human photoreceptors. Some interesting IOSs have already been detected in inner retinal layers, but better development of the IOS instruments and software algorithms is required to achieve optimal physiological assessment of inner retinal neurons.

We have investigated Čerenkov radiation generated in phosphor-based optical fiber dosimeters irradiated with clinical electron beams. We fabricated two high-spatial resolution fiber-optic probes, with 200 and 400  μm core diameters, composed of terbium-based phosphor tips. A generalizable spectroscopic method was used to separate Čerenkov radiation from the transmitted signal by the fiber based on the assumption that the recorded signal is a linear superposition of two basis spectra: characteristic luminescence of the phosphor medium and Čerenkov radiation. We performed Monte Carlo simulations of the Čerenkov radiation generated in the fiber and found a strong dependence of the recorded Čerenkov radiation on the numerical aperture of the fiber at shallow phantom depths; however, beyond the depth of maximum dose that dependency is minimal. The simulation results agree with the experimental results for Čerenkov radiation generated in fibers. The spectroscopic technique used in this work can be used for development of high-spatial resolution fiber micro dosimeters and for optical characterization of various scintillating materials, such as phosphor nanoparticles, in ionizing radiation fields of high energy.

Anemia is a prevalent medical condition that seriously affects millions of people all over the world. In many regions, not only its initial detection but also its monitoring are hindered by limited access to laboratory facilities. This situation has motivated the development of a wide range of optical devices and procedures to assist physicians in these tasks. Although noticeable progress has been achieved in this area, the search for reliable, low-cost, and risk-free solutions still continues, and the strengthening of the knowledge base about this disorder and its effects is essential for the success of these initiatives. We contribute to these efforts by closely examining the sensitivity of human skin hyperspectral responses (within and outside the visible region of the light spectrum) to reduced hemoglobin concentrations associated with increasing anemia severity levels. This investigation, which involves skin specimens with distinct biophysical and morphological characteristics, is supported by controlled in silico experiments performed using a predictive light transport model and measured data reported in the biomedical literature. We also propose a noninvasive procedure to be employed in the monitoring of this condition at the point-of-care.

Resonance enhancement of Raman spectroscopy (RS) has been used to significantly improve the sensitivity and selectivity of detection for specific components in complicated environments. Resonance RS gives more insight into the biochemical structure and reactivity. In this field, selecting a proper excitation wavelength to achieve optimal resonance enhancement is vital for the study of an individual chemical/biological ingredient with a particular absorption characteristic. Raman spectra of three azo derivatives with absorption spectra in the visible range are studied under the same experimental conditions at 488, 532, and 633 nm excitations. Universal laws in the visible range have been concluded by analyzing resonance Raman (RR) spectra of samples. The long wavelength edge of the absorption spectrum is a better choice for intense enhancement and the integrity of a Raman signal. The obtained results are valuable for applying RR for the selective detection of biochemical constituents whose electronic transitions take place at energies corresponding to the visible spectra, which is much friendlier to biologial samples compared to ultraviolet.

Reflectance confocal microscopy is successfully used in infant skin research. Infant skin structure, function, and composition are undergoing a maturation process. We aimed to uncover how the epidermal architecture and cellular topology change with time. Images were collected from three age groups of healthy infants between one and four years of age and adults. Cell centers were manually identified on the images at the stratum granulosum (SG) and stratum spinosum (SS) levels. Voronoi diagrams were used to calculate geometrical and topological parameters. Infant cell density is higher than that of adults and decreases with age. Projected cell area, cell perimeter, and average distance to the nearest neighbors increase with age but do so distinctly between the two layers. Structural entropy is different between the two strata, but remains constant with time. For all ages and layers, the distribution of the number of nearest neighbors is typical of a cooperator network architecture. The topological analysis provides evidence of the maturation process in infant skin. The differences between infant and adult are more pronounced in the SG than SS, while cell cooperation is evident in all cases of healthy skin examined.

A recent study showed that 355-nm nanosecond lasers cut cornea with similar precision to infrared femtosecond lasers. However, use of ultraviolet wavelength requires precise assessment of ocular safety to determine the range of possible ophthalmic applications. In this study, the 355-nm nanosecond laser was evaluated for corneal and iris damage in rabbit, porcine, and human donor eyes as determined by minimum visible lesion (MVL) observation, live/dead staining of the endothelium, and apoptosis assay. Single-pulse damage to the iris was evaluated on porcine eyes using live/dead staining. In live rabbits, the cumulative median effective dose (ED50) for corneal damage was 231  J  /  cm2, as seen by lesion observation. Appearance of endothelial damage in live/dead staining or apoptosis occurred at higher radiant exposure of 287  J  /  cm2. On enucleated rabbit and porcine corneas, ED50 was 87 and 52  J  /  cm2, respectively, by MVL, and 241 and 160  J  /  cm2 for endothelial damage. In human eyes, ED50 for MVL was 110  J  /  cm2 and endothelial damage at 453  J  /  cm2. Single-pulse iris damage occurred at ED50 of 208  mJ  /  cm2. These values determine the energy permitted for surgical patterns and can guide development of ophthalmic laser systems. Lower damage threshold in corneas of enucleated eyes versus live rabbits is noted for future safety evaluation.

A modified spatial-temporal speckle correlation technique for operational assessment of structural changes in muscle tissues after slaughtering is considered. Coefficient of biological activity as a quantitative indicator of structural changes of biochemical processes in biological tissues is proposed. The experimental results have shown that this coefficient properly evaluates the biological activity of pig and chicken muscle tissue samples. Studying the degradation processes in muscle tissue during long-time storage in a refrigerator by measuring the spatial-temporal dynamics of biospeckle patterns is carried out. The reduction of the bioactivity level of refrigerated muscle tissue samples connected with the initiation of muscle fiber cracks and ruptures, reduction of sarcomeres, nuclei deformation, nuclear chromatin diminishing, and destruction of mitochondria is analyzed.

A noncontact optical detector for in vivo imaging has been developed that is compatible with magnetic resonance imaging (MRI). The optical detector employs microlens arrays and might be classified as a plenoptic camera. As a resulting of its design, the detector possesses a slim thickness and is self-shielding against radio frequency (RF) pulses. For experimental investigation, a total of six optical detectors were arranged in a cylindrical fashion, with the imaged object positioned in the center of this assembly. A purposely designed RF volume resonator coil has been developed and is incorporated within the optical imaging system. The whole assembly was placed into the bore of a 1.5 T patient-sized MRI scanner. Simple-geometry phantom studies were performed to assess compatibility and performance characteristics regarding both optical and MR imaging systems. A bimodal ex vivo nude mouse measurement was conducted. From the MRI data, the subject surface was extracted. Optical images were projected on this surface by means of an inverse mapping algorithm. Simultaneous measurements did not reveal influences from the magnetic field and RF pulses onto optical detector performance (spatial resolution, sensitivity). No significant influence of the optical imaging system onto MRI performance was detectable.

Skin moisture relates to the state of multiple skin constituents and aspects, but unfortunately, a device which could provide comprehensive and in vivo analysis is not available. Nevertheless, several reports have demonstrated accurate estimations of dermal water content using near-infrared spectroscopy (NIRS), and the potential of employing this technique in skin analysis. We aim to investigate whether NIRS could detect changes in skin barrier function through evaluation of skin water uptake in relation to moisturizer application. NIR and capacitance data were collected from 20 volunteers at both forearms, prior to and after seven days of regular moisturizer use. Results indicated lower peak intensities at the 1940-nm minima and higher intensities at the 1450-nm equivalent minima with moisturizer abstinence, while the opposite was true with regular moisturizer application. As the light beam would have traveled deeper into the skin at 1450 nm, it has been concluded that long-term, frequent moisturizer use had limited the penetration of extrinsic water. Partial least squares analysis showed that separation of sample’s scores increased with abstinence of moisturizer use. Thus, NIRS can provide valuable information not only on dermal water contents but also on additional parameters such as skin barrier function.

A terahertz (THz) spectroscopic study is carried out to analyze DNA mutations in a label-free manner. Three newly designed liquid sample cells are considered and the best is selected as the sample carrier for THz transmission spectroscopic analyses. Discrimination based on spectral signatures of single-base mutations on single-stranded 20 nt oligonucleotides has been shown possible experimentally. The results clearly attest the ability of this promising approach for label-free analyses of single-base mutations of DNA molecules. This study has demonstrated that the THz spectroscopic technology can be considered as a potential diagnostic tool for investigating molecular reactions, such as DNA mutations.

Fluorescence microscopy is commonly used to investigate disease progression in biological tissues. Biological tissues, however, are strongly scattering in the visible wavelengths, limiting the application of fluorescence microscopy to superficial (<200  μm) regions. Optical clearing, which involves incubation of the tissue in a chemical bath, reduces the optical scattering in tissue, resulting in increased tissue transparency and optical imaging depth. The goal of this study was to determine the time- and wavelength-resolved dynamics of the optical scattering properties of rodent brain after optical clearing with FocusClear™. Light transmittance and reflectance of 1-mm mouse brain sections were measured using an integrating sphere before and after optical clearing and the inverse adding doubling algorithm used to determine tissue optical scattering. The degree of optical clearing was quantified by calculating the optical clearing potential (OCP), and the effects of differing OCP were demonstrated using the optical histology method, which combines tissue optical clearing with optical imaging to visualize the microvasculature. We observed increased tissue transparency with longer optical clearing time and an analogous increase in OCP. Furthermore, OCP did not vary substantially between 400 and 1000 nm for increasing optical clearing durations, suggesting that optical histology can improve ex vivo visualization of several fluorescent probes.

Thresholds for microcavitation of bovine and porcine melanosomes were previously reported, using single nanosecond (ns) laser pulses in the visible (532 nm) and the near-infrared (NIR) from 1000 to 1319 nm. Here, we report average radiant exposure thresholds for bovine melanosome microcavitation at additional NIR wavelengths up to 1540 nm, which range from ∼0.159  J/cm2 at 800 nm to 4.5  J/cm2 at 1540 nm. Melanosome absorption coefficients were also estimated, and decreased with increasing wavelength. These values were compared to retinal pigment epithelium coefficients, and to water absorption, over the same wavelength range. Corneal total intraocular energy retinal damage threshold values were estimated and compared to the previous (2007) and recently changed (2014) maximum permissible exposure (MPE) safe levels. Results provide additional data that support the recent changes to the MPE levels, as well as the first microcavitation data at 1540 nm, a wavelength for which melanosome microcavitation may be an ns-pulse skin damage mechanism.

Ultrasmall scale implants have recently attracted focus as valuable tools for monitoring both acute and chronic diseases. Semiconductor optical technologies are the key to miniaturizing these devices to the long-sought sub-mm scale, which will enable long-term use of these devices for medical applications. This can also enable the use of multiple implantable devices concurrently to form a true body area network of sensors. We demonstrate optical power transfer techniques and methods to effectively harness this power for implantable devices. Furthermore, we also present methods for optical data transfer from such implants. Simultaneous use of these technologies can result in miniaturized sensing platforms that can allow for large-scale use of such systems in real world applications.

Lipid-correlated disease such as atherosclerosis has been an important medical research topic for decades. Many new microscopic imaging techniques such as coherent anti-Stokes Raman scattering and third-harmonic generation (THG) microscopy were verified to have the capability to target lipids in vivo. In the case of THG microscopy, biological cell membranes and lipid bodies in cells and tissues have been shown as good sources of contrast with a laser excitation wavelength around 1200 nm. We report the THG excitation spectroscopy study of two pure free fatty acids including oleic acid and linoleic acid from 1090 to 1330 nm. Different pure fatty acids presented slightly-different THG χ(3) spectra. The measured peak values of THG third-order susceptibility χ(3) in both fatty acids were surprisingly found not to match completely with the resonant absorption wavelengths around 1190 to 1210 nm, suggesting possible wavelengths selection for enhanced THG imaging of lipids while avoiding laser light absorption. Along with the recent advancement in THG imaging, this new window between 1240 to 1290 nm may offer tremendous new opportunities for sensitive label-free lipid imaging in biological tissues.

The voxel-based Monte Carlo method (VMC) is now a gold standard in the simulation of light propagation in turbid media. For complex tissue structures, however, the computational cost will be higher when small voxels are used to improve smoothness of tissue interface and a large number of photons are used to obtain accurate results. To reduce computational cost, criteria were proposed to determine the voxel size and photon number in 3-dimensional VMC simulations with acceptable accuracy and computation time. The selection of the voxel size can be expressed as a function of tissue geometry and optical properties. The photon number should be at least 5 times the total voxel number. These criteria are further applied in developing a photon ray splitting scheme of local grid refinement technique to reduce computational cost of a nonuniform tissue structure with significantly varying optical properties. In the proposed technique, a nonuniform refined grid system is used, where fine grids are used for the tissue with high absorption and complex geometry, and coarse grids are used for the other part. In this technique, the total photon number is selected based on the voxel size of the coarse grid. Furthermore, the photon-splitting scheme is developed to satisfy the statistical accuracy requirement for the dense grid area. Result shows that local grid refinement technique photon ray splitting scheme can accelerate the computation by 7.6 times (reduce time consumption from 17.5 to 2.3 h) in the simulation of laser light energy deposition in skin tissue that contains port wine stain lesions.

Bioluminescence imaging is a noninvasive technique whereby surface weighted images of luminescent probes within animals are used to characterize cell count and function. Traditionally, data are collected over the entire emission spectrum of the source using no filters and are used to evaluate cell count/function over the entire spectrum. Alternatively, multispectral data over several wavelengths can be incorporated to perform tomographic reconstruction of source location and intensity. However, bandpass filters used for multispectral data acquisition have a specific bandwidth, which is ignored in the reconstruction. In this work, ignoring the bandwidth is shown to introduce a dependence of the recovered source intensity on the bandwidth of the filters. A method of accounting for the bandwidth of filters used during multispectral data acquisition is presented and its efficacy in increasing the quantitative accuracy of bioluminescence tomography is demonstrated through simulation and experiment. It is demonstrated that while using filters with a large bandwidth can dramatically decrease the data acquisition time, if not accounted for, errors of up to 200% in quantitative accuracy are introduced in two-dimensional planar imaging, even after normalization. For tomographic imaging, the use of this method to account for filter bandwidth dramatically improves the quantitative accuracy.

Spectrally resolved confocal microscopy and fluorescence lifetime imaging have been used to measure the polarity of lipid-rich regions in living HeLa cells stained with Nile red. The emission peak from the solvatochromic dye in lipid droplets is at a shorter wavelength than other, more polar, stained internal membranes, and this is indicative of a low polarity environment. We estimate that the dielectric constant, ϵ, is around 5 in lipid droplets and 25<ϵ<40 in other lipid-rich regions. Our spectrally resolved fluorescence lifetime imaging microscopy (FLIM) data show that intracellular Nile red exhibits complex, multiexponential fluorescence decays due to emission from a short lifetime locally excited state and a longer lifetime intramolecular charge transfer state. We measure an increase in the average fluorescence lifetime of the dye with increasing emission wavelength, as shown using phasor plots of the FLIM data. We also show using these phasor plots that the shortest lifetime decay components arise from lipid droplets. Thus, fluorescence lifetime is a viable contrast parameter for distinguishing lipid droplets from other stained lipid-rich regions. Finally, we discuss the FLIM of Nile red as a method for simultaneously mapping both polarity and relative viscosity based on fluorescence lifetime measurements.

Fluorescence lifetime imaging microscopy (FLIM) is a microscopic imaging technique to present an image of fluorophore lifetimes. It circumvents the problems of typical imaging methods such as intensity attenuation from depth since a lifetime is independent of the excitation intensity or fluorophore concentration. The lifetime is estimated from the time sequence of photon counts observed with signal-dependent noise, which has a Poisson distribution. Conventional methods usually estimate single or biexponential decay parameters. However, a lifetime component has a distribution or width, because the lifetime depends on macromolecular conformation or inhomogeneity. We present a novel algorithm based on a sparse representation which can estimate the distribution of lifetime. We verify the enhanced performance through simulations and experiments.

Vitrification is an increasingly popular method of embryo cryopreservation that is used in assisted reproductive technology. Although vitrification has high post-thaw survival rates compared to other freezing techniques, its long-term effects on embryo development are still poorly understood. We demonstrate an application of full-field optical coherence tomography (FF-OCT) to visualize the effects of vitrification on live single-cell (2 pronuclear) mouse embryos without harmful labels. Using FF-OCT, we observed that vitrification causes a significant increase in the aggregation of structures within the embryo cytoplasm, consistent with reports in literature based on fluorescence techniques. We quantify the degree of aggregation with an objective metric, the cytoplasmic aggregation (CA) score, and observe a high degree of correlation between the CA scores of FF-OCT images of embryos and of fluorescence images of their mitochondria. Our results indicate that FF-OCT shows promise as a label-free assessment of the effects of vitrification on embryo mitochondria distribution. The CA score provides a quantitative metric to describe the degree to which embryos have been affected by vitrification and could aid clinicians in selecting embryos for transfer.

The distribution of refractive indices (RIs) of a living cell contributes in a nonintuitive manner to its optical phase image and quite rarely can be inverted to recover its internal structure. The interpretation of the quantitative phase images of living cells remains a difficult task because (1) we still have very little knowledge on the impact of its internal macromolecular complexes on the local RI and (2) phase changes produced by light propagation through the sample are mixed with diffraction effects by the internal cell bodies. We propose to implement a two-dimensional wavelet-based contour chain detection method to distinguish internal boundaries based on their greatest optical path difference gradients. These contour chains correspond to the highest image phase contrast and follow the local RI inhomogeneities linked to the intracellular structural intricacy. Their statistics and spatial distribution are the morphological indicators suited for comparing cells of different origins and/or to follow their transformation in pathologic situations. We use this method to compare nonadherent blood cells from primary and laboratory culture origins and to assess the internal transformation of hematopoietic stem cells by the transduction of the BCR-ABL oncogene responsible for the chronic myelogenous leukemia.

Photoacoustic imaging (PAI) contributes to tumor diagnosis through the use of PAI probes that effectively accumulate in tumors. Previously, we developed a symmetrical cyanine dye, IC7-1-Bu, which showed high potential as a PAI probe because of its high tumor targeting ability and sufficient in vivo PA signal. However, IC7-1-Bu lacks photostability for multiple laser irradiations, so we developed stabilized PAI probes using IC7-1-Bu as a lead compound. We focused on the effect of singlet oxygen (O12) generated by excited PAI probes on probe degeneration. We introduced a triplet-state quencher (TSQ) moiety into IC7-1-Bu to quench O12 generation and designed three IC-n-T derivatives with different linker lengths (n indicates linker length). The IC-n-T derivatives emitted in vitro PA signals that were comparable to IC7-1-Bu and significantly reduced O12 generation while showing improved photostability against multiple irradiations. Of the three derivatives evaluated, IC-5-T accumulated in tumors effectively to allow clear PAI of tumors in vivo. Furthermore, the photostability of IC-5-T was 1.5-fold higher than that of IC7-1-Bu in in vivo sequential PAI. These results suggest that IC-5-T is a potential PAI probe for in vivo sequential tumor imaging.

Intraductal carcinoma is a precancerous lesion of the breast and the immediate precursor of invasive ductal carcinoma. Multiphoton microscopy (MPM) was used to monitor the progression from intraductal carcinoma to invasive ductal carcinoma, which can improve early detection of precursor lesions and halt progression to invasive neoplastic disease. It was found that MPM has the capability to reveal the qualitative changes in features of cells, structure of basement membranes, and architecture of collagens during the development from intraductal carcinoma to invasive ductal carcinoma, as well as the quantitative alterations in nuclear area, circle length of basement membrane, and collagen density. Combined with intra-fiberoptic ductoscopy or transdermal biopsy needle, MPM has the potential to provide immediate histological diagnosis of tumor progression in the field of breast carcinoma.

Subarachnoid hemorrhage (SAH) is often accompanied by cerebral vasospasm (CVS), which is the phenomenon of narrowing of large cerebral arteries, and then can produce delayed ischemic neurological deficit (DIND) such as lateralized sensory dysfunction. CVS was regarded as a major contributor to DIND in patients with SAH. However, therapy for preventing vasospasm after SAH to improve the outcomes may not work all the time. It is important to find answers to the relationship between CVS and DIND after SAH. How local cerebral blood flow (CBF) is regulated during functional activation after SAH still remains poorly understood, whereas, the regulation of CBF may play an important role in weakening the impact of CVS on cortex function. Therefore, it is worthwhile to evaluate the functional response of CBF in the activated cortex in an SAH animal model. Most evaluation of the effect of SAH is presently carried out by neurological behavioral scales. The functional imaging of cortical activation during sensory stimulation may help to reflect the function of the somatosensory cortex more locally than the behavioral scales do. We investigated the functional response of CBF in the somatosensory cortex induced by an electrical stimulation to contralateral forepaw via laser speckle imaging in a rat SAH model. Nineteen Sprague-Dawley rats from two groups (control group, n=10 and SAH group, n=9) were studied. SAH was induced in rats by double injection of autologous blood into the cisterna magna after CSF aspiration. The same surgical procedure was applied in the control group without CSF aspiration or blood injection. Significant CVS was found in the SAH group. Meanwhile, we observed a delayed peak of CBF response in rats with SAH compared with those in the control group, whereas no significant difference was found in magnitude, duration, and areas under curve of relative CBF changes between the two groups. The results suggest that the regulation function of local CBF during functional activation induced by somatosensory stimulation might not be seriously impaired in the somatosensory cortex of rats with SAH. Therefore, our findings might help to understand the clinical phenomenon that DIND might not occur even when CVS was found in SAH patients.

Reconstructing a three-dimensional scene from multiple simultaneously acquired perspectives (the light field) is an elegant scanless imaging concept that can exceed the temporal resolution of currently available scanning-based imaging methods for capturing fast cellular processes. We tested the performance of commercially available light field cameras on a fluorescent microscopy setup for monitoring calcium activity in the brain of awake and behaving reporter zebrafish larvae. The plenoptic imaging system could volumetrically resolve diverse neuronal response profiles throughout the zebrafish brain upon stimulation with an aversive odorant. Behavioral responses of the reporter fish could be captured simultaneously together with depth-resolved neuronal activity. Overall, our assessment showed that with some optimizations for fluorescence microscopy applications, commercial light field cameras have the potential of becoming an attractive alternative to custom-built systems to accelerate molecular imaging research on cellular dynamics.

A wearable all-plastic imaging system for real-time fluorescence image-guided surgery is presented. The compact size of the system is especially suitable for applications in the operating room. The system consists of a dual-mode imaging system, see-through goggle, autofocusing, and auto-contrast tuning modules. The paper will discuss the system design and demonstrate the system performance.

Rheumatoid arthritis (RA) is a disease that frequently leads to joint destruction. It has a high incidence rate worldwide, and the disease significantly reduces patients’ quality of life. Detecting and treating inflammatory arthritis before structural damage to the joint has occurred is known to be essential for preventing patient disability and pain. Existing diagnostic technologies are expensive, time consuming, and require trained personnel to collect and interpret data. Optical techniques might be a fast, noninvasive alternative. Hyperspectral imaging (HSI) is a noncontact optical technique which provides both spectral and spatial information in one measurement. In this study, the feasibility of HSI in arthritis diagnostics was explored by numerical simulations and optimal imaging parameters were identified. Hyperspectral reflectance and transmission images of RA and normal human joint models were simulated using the Monte Carlo method. The spectral range was 600 to 1100 nm. Characteristic spatial patterns for RA joints and two spectral windows with transmission were identified. The study demonstrated that transmittance images of human joints could be used as one parameter for discrimination between arthritic and unaffected joints. The presented work shows that HSI is a promising imaging modality for the diagnostics and follow-up monitoring of arthritis in small joints.

Rodent middle cerebral artery occlusion (MCAO) model is commonly used in stroke research. Creating a stable infarct volume has always been challenging for technicians due to the variances of animal anatomy and surgical operations. The depth of filament suture advancement strongly influences the infarct volume as well. We investigated the cerebral blood flow (CBF) changes in the affected cortex using laser speckle contrast imaging when advancing suture during MCAO surgery. The relative CBF drop area (CBF50, i.e., the percentage area with CBF less than 50% of the baseline) showed an increase from 20.9% to 69.1% when the insertion depth increased from 1.6 to 1.8 cm. Using the real-time CBF50 marker to guide suture insertion during the surgery, our animal experiments showed that intraoperative CBF-guided surgery could significantly improve the stability of MCAO with a more consistent infarct volume and less mortality.

The growing interest in performing high-resolution, deep-tissue imaging has galvanized the use of longer excitation wavelengths and three-photon-based techniques in nonlinear imaging modalities. This study presents a threefold improvement in maximum imaging depth of ex vivo porcine vocal folds using third-harmonic generation (THG) microscopy at 1552-nm excitation wavelength compared to two-photon microscopy (TPM) at 776-nm excitation wavelength. The experimental, analytical, and Monte Carlo simulation results reveal that THG improves the maximum imaging depth observed in TPM significantly from 140 to 420  μm in a highly scattered medium, reaching the expected theoretical imaging depth of seven extinction lengths. This value almost doubles the previously reported normalized imaging depths of 3.5 to 4.5 extinction lengths using three-photon-based imaging modalities. Since tissue absorption is substantial at the excitation wavelength of 1552 nm, this study assesses the tissue thermal damage during imaging by obtaining the depth-resolved temperature distribution through a numerical simulation incorporating an experimentally obtained thermal relaxation time (τ). By shuttering the laser for a period of 2τ, the numerical algorithm estimates a maximum temperature increase of ∼2°C at the maximum imaging depth of 420  μm. The paper demonstrates that THG imaging using 1552 nm as an illumination wavelength with effective thermal management proves to be a powerful deep imaging modality for highly scattering and absorbing tissues, such as scarred vocal folds.

An approach to segment macular layer thicknesses from spectral domain optical coherence tomography has been proposed. The main contribution is to decrease computational costs while maintaining high accuracy via exploring Kalman filtering, customized active contour, and curve smoothing. Validation on 21 normal volumes shows that 8 layer boundaries could be segmented within 5.8 s with an average layer boundary error <2.35  μm. It has been compared with state-of-the-art methods for both normal and age-related macular degeneration cases to yield similar or significantly better accuracy and is 37 times faster. The proposed method could be a potential tool to clinically quantify the retinal layer boundaries.

Optical coherence tomography/laser induced fluorescence (OCT/LIF) dual-modality imaging allows for minimally invasive, nondestructive endoscopic visualization of colorectal cancer in mice. This technology enables simultaneous longitudinal tracking of morphological (OCT) and biochemical (fluorescence) changes as colorectal cancer develops, compared to current methods of colorectal cancer screening in humans that rely on morphological changes alone. We have shown that QDot655 targeted to vascular endothelial growth factor receptor 2 (QD655-VEGFR2) can be applied to the colon of carcinogen-treated mice and provides significantly increased contrast between the diseased and undiseased tissue with high sensitivity and specificity ex vivo. QD655-VEGFR2 was used in a longitudinal in vivo study to investigate the ability to correlate fluorescence signal to tumor development. QD655-VEGFR2 was applied to the colon of azoxymethane (AOM-) or saline-treated control mice in vivo via lavage. OCT/LIF images of the distal colon were taken at five consecutive time points every three weeks after the final AOM injection. Difficulties in fully flushing unbound contrast agent from the colon led to variable background signal; however, a spatial correlation was found between tumors identified in OCT images, and high fluorescence intensity of the QD655 signal, demonstrating the ability to detect VEGFR2 expressing tumors in vivo.

Fluorescence-based single molecule techniques to interrogate gene expression in tissues present a very low signal-to-noise ratio due to the strong autofluorescence and other background signals from tissue sections. This report presents a background-free method using second-harmonic generation (SHG) nanocrystals as probes to quantify the messenger RNA (mRNA) of human epidermal growth receptor 2 (Her2) at single molecule resolution in specific phenotypes at single-cell resolution directly in tissues. Coherent SHG emission from individual barium titanium oxide (BTO) nanoprobes was demonstrated, allowing for a stable signal beyond the autofluorescence window. Her2 surface marker and Her2 mRNA were specifically labeled with BTO probes, and Her2 mRNA was quantified at single copy sensitivity in Her2 expressing phenotypes directly in cancer tissues. Our approach provides the first proof of concept of a cross-platform strategy to probe tissues at single-cell resolution in situ.

Conventional and dark-field microscopy in the transmission mode is extensively used for single plasmonic nanoparticle (NP) imaging and spectral analysis. However, application of the transmission mode for real-time biosensing to single NP poses strict limitations on the size and material properties of the microfluidic system. This article proposes a simple optical technique based on reflected light microscopy to perform microspectroscopy of a single NP placed in a conventional, nontransparent liquid delivery system. The insertion of a variable spot diaphragm in the optical path reduces the interference effect that occurs at the NP-substrate interface and improves the signal-to-noise ratio in NP imaging. Using this method, we demonstrated spatial imaging and spectral analyses of 60-, 80-, and 100-nm single gold NPs. A single-NP sensor based on a 100-nm NP was used for real-time measurement of bulk refractive index changes in the microfluidic channel and for detection of fast dynamic poly(ethylene glycol) attachment to the NP surface. Finally, electrochemical single-particle microspectroscopy was demonstrated by using a methylene blue electroactive redox tag. The proposed optical approach is expected to significantly improve the miniaturization and multiplexing capabilities of high-throughput biosensing based on single NP.

Reflectance measurements acquired from within the subdiffusion regime (i.e., lengthscales smaller than a transport mean free path) retain much of the original information about the shape of the scattering phase function. Given this sensitivity, many models of subdiffusion regime light propagation have focused on parametrizing the optical signal through various optical and empirical parameters. We argue, however, that a more useful and universal way to characterize such measurements is to focus instead on the fundamental physical properties, which give rise to the optical signal. This work presents the methodologies that used to model and extract tissue ultrastructural and microvascular properties from spatially resolved subdiffusion reflectance spectroscopy measurements. We demonstrate this approach using ex-vivo rat tissue samples measured by enhanced backscattering spectroscopy.

The aim of the present study was to assess the effect of low-level laser therapy (LLLT) in the treatment of burning mouth syndrome (BMS). A diode laser was used in 78 BMS patients who were randomly assigned into four groups: IR1W, n=20 (830 nm, 100 mW, 5 J, 176  J/cm2, 50 s, LLLT weekly sessions, 10 sessions); IR3W, n=20 (830 nm, 100 mW, 5 J, 176  J/cm2, 50 s, three LLLT weekly sessions, 9 sessions); red laser, n=19 (685 nm, 35 mW, 2 J, 72  J/cm2, 58 s, three LLLT weekly sessions, 9 sessions); and control-group (CG), n=19. Symptoms were assessed at the end of the treatment and eight weeks later; quality of life related to oral health was assessed using the Oral Health Impact Profile (OHIP-14). Statistical analysis was carried out using repeated measures analysis of variance followed by the posthoc Tukey test. There was significant reduction of the symptoms in all groups at the end of the treatment, which was maintained in the follow-up. The scores of the IR1W and IR3W laser groups differed significantly from those of the CG. There was also a decrease in the OHIP-14 scores in the four groups. The IR3W laser group scores differed significantly from those of the CG. LLLT reduces the symptoms of BMS and may be an alternative therapeutic strategy for the relief of symptoms in patients with BMS.

Protoporphyrin IX (PPIX) produced following the administration of exogenous 5d-aminolevulinic acid is clinically approved for photodynamic therapy and fluorescence-guided resection in various jurisdictions around the world. For both applications, quantification of PPIX forms the basis for accurate therapeutic dose calculation and identification of malignant tissues for resection. While it is well established that the PPIX synthesis and accumulation rates are subject to the cell’s biochemical microenvironment, the effect of the physical microenvironment, such as matrix stiffness, has received little attention to date. Here we studied the proliferation rate and PPIX synthesis and accumulation in two glioma cell lines U373 and U118 cultured under five different substrate conditions, including the conventional tissue culture plastic and polyacrylamide gels that simulated tissue stiffness of normal brain (1 kPa) and glioblastoma tumors (12 kPa). We found that the proliferation rate increased with substrate stiffness for both cell lines, but not in a linear fashion. PPIX concentration was significantly higher in cells cultured on tissue-simulating gels than on the much stiffer tissue culture plastic for both cell lines. These findings, albeit preliminary, suggest that the physical microenvironment might be an important determinant of tumor aggressiveness and PPIX synthesis in glioma cells.