Angiogenesis in a tumor region creates arteriovenous (AV) shunts that cause an abnormal venous blood oxygen saturation (sO₂) distribution. Here, we applied optical-resolution photoacoustic microscopy to study the AV shunting in vivo. First, we built a phantom to image sO₂ distribution in a vessel containing converged flows from two upstream blood vessels with different sO₂ values. The phantom experiment showed that the blood from the two upstream vessels maintained a clear sO₂ boundary for hundreds of seconds, which is consistent with our theoretical analysis using a diffusion model. Next, we xenotransplanted O-786 tumor cells in mouse ears and observed abnormal sO₂ distribution in the downstream vein from the AV shunts in vivo. Finally, we identified the tumor location by tracing the sO₂ distribution. Our study suggests that abnormal sO₂ distribution induced by the AV shunts in the vessel network may be used as a new functional benchmark for early tumor detection.

Recently, optical coherence tomography (OCT) angiography has enabled label-free imaging of vasculature based on dynamic scattering in vessels. However, quantitative volumetric analysis of the vascular networks depicted in OCT angiography data has remained challenging. Multiple-scattering tails (artifacts specific to the imaging geometry) make automated assessment of vascular morphology problematic. We demonstrate that dynamically focused optical coherence microscopy (OCM) angiography with a high numerical aperture, chosen so the scattering length greatly exceeds the depth-of-field, significantly reduces the deleterious effect of multiple-scattering tails in synthesized angiograms. Capitalizing on the improved vascular image quality, we devised and tailored a self-correcting automated graphing approach that achieves a reconstruction of cortical microvasculature from OCM angiography data sets with accuracy approaching that attained by trained operators. The automated techniques described here will facilitate more widespread study of vascular network topology in health and disease.

A multimodality Raman and photoacoustic imaging system is presented. This system has ultralow background and can detect tumor cells labeled with modified surface-enhanced-Raman-scattering (SERS) nanoparticles in vivo. Photoacoustic imaging provides microvascular context and can potentially be used to guide magnetic trapping of circulating tumor cells for SERS detection in animal models.

In vivo endoscopic Raman spectroscopy of human tissue using a fiber optic probe has been previously demonstrated. However, there remain several technical challenges, such as a robust control over the laser radiation dose and measurement repeatability during endoscopy. A decrease in the signal to noise was also observed due to aging of Raman probe after repeated cycles of harsh reprocessing procedures. To address these issues, we designed and tested a disposable, biocompatible, and sterile sheath for use with a fiber optic endoscopic Raman probe. The sheath effectively controls contamination of Raman probes between procedures, greatly reduces turnaround time, and slows down the aging of the Raman probes. A small optical window fitted at the sheath cap maintained the measurement distance between Raman probe end and tissue surface. To ensure that the sheath caused a minimal amount of fluorescence and Raman interference, the optical properties of materials for the sheath, optical window, and bonding agent were studied. The easy-to-use sheath can be manufactured at a moderate cost. The sheath strictly enforced a maximum permissible exposure standard of the tissue by the laser and reduced the spectral variability by 1.5 to 8.5 times within the spectral measurement range.

We describe the development of an optical flow visualization method for resolving the flow velocity vector field in lymphatic vessels in vitro. The aim is to develop an experimental protocol for accurately estimating flow parameters, such as flow rate and shear stresses, with high spatial and temporal resolution. Previous studies in situ have relied on lymphocytes as tracers, but their low density resulted in a reduced spatial resolution whereas the assumption that the flow was fully developed in order to determine the flow parameters of interest may not be valid, especially in the vicinity of the valves, where the flow is undoubtedly more complex. To overcome these issues, we have applied the time-resolved microparticle image velocimetry (μ-PIV) technique, a well-established method that can provide increased spatial and temporal resolution that this transient flow demands. To that end, we have developed a custom light source, utilizing high-power light-emitting diodes, and associated control and image processing software. This paper reports the performance of the system and the results of a series of preliminary experiments performed on vessels isolated from rat mesenteries, demonstrating, for the first time, the successful application of the μ-PIV technique in these vessels.

Sound processing in the inner ear involves separation of the constituent frequencies along the length of the cochlea. Frequencies relevant to human speech (100 to 500 Hz) are processed in the apex region. Among mammals, the guinea pig cochlear apex processes similar frequencies and is thus relevant for the study of speech processing in the cochlea. However, the requirement for extensive surgery has challenged the optical accessibility of this area to investigate cochlear processing of signals without significant intrusion. A simple method is developed to provide optical access to the guinea pig cochlear apex in two directions with minimal surgery. Furthermore, all prior vibration measurements in the guinea pig apex involved opening an observation hole in the otic capsule, which has been questioned on the basis of the resulting changes to cochlear hydrodynamics. Here, this limitation is overcome by measuring the vibrations through the unopened otic capsule using phase-sensitive Fourier domain optical coherence tomography. The optically and surgically advanced method described here lays the foundation to perform minimally invasive investigation of speech-related signal processing in the cochlea.

The noninvasive assessment of flap viability in autologous reconstruction surgery is still an unmet clinical need. To cope with this problem, we developed a proof-of-principle fully automatized setup for fast time-gated diffuse optical tomography exploiting Mellin–Laplace transform to obtain three-dimensional tomographic reconstructions of oxy- and deoxy-hemoglobin concentrations. We applied this method to perform preclinical tests on rats inducing total venous occlusion in the cutaneous abdominal flaps. Notwithstanding the use of just four source-detector couples, we could detect a spatially localized increase of deoxyhemoglobin following the occlusion (up to 550  μM in 54 min). Such capability to image spatio-temporal evolution of blood perfusion is a key issue for the noninvasive monitoring of flap viability.

The health of the ocular surface requires blinks of the eye to be frequent in order to provide moisture and to renew the tear film. However, blinking frequency has been shown to decrease in certain conditions such as when subjects are conducting tasks with high cognitive and visual demands. These conditions are becoming more common as people work or spend their leisure time in front of video display terminals. Supervision of blinking frequency in such environments is possible, thanks to the availability of computer-integrated cameras. Therefore, the aim of the present study is to develop an algorithm for the detection of eye blinks and to test it, in a number of videos captured, while subjects are conducting a variety of tasks in front of the computer. The sensitivity of the algorithm for blink detection was found to be of 87.54% (range 30% to 100%), with a mean false-positive rate of 0.19% (range 0% to 1.7%), depending on the illumination conditions during which the image was captured and other computer–user spatial configurations. The current automatic process is based on a partly modified pre-existing eye detection and image processing algorithms and consists of four stages that are aimed at eye detection, eye tracking, iris detection and segmentation, and iris height/width ratio assessment.

Urinary bladder diseases are a common problem throughout the world and often difficult to accurately diagnose. Furthermore, they pose a heavy financial burden on health services. Urinary bladder tissue from male pigs was spectrophotometrically measured and the resulting data used to calculate the absorption, transmission, and reflectance parameters, along with the derived coefficients of scattering and absorption. These were employed to create a “generic” computational bladder model based on optical properties, simulating the propagation of photons through the tissue at different wavelengths. Using the Monte-Carlo method and fluorescence spectra of UV and blue excited wavelength, diagnostically important biomarkers were modeled. Additionally, the multifunctional noninvasive diagnostics system “LAKK-M” was used to gather fluorescence data to further provide essential comparisons. The ultimate goal of the study was to successfully simulate the effects of varying excited radiation wavelengths on bladder tissue to determine the effectiveness of photonics diagnostic devices. With increased accuracy, this model could be used to reliably aid in differentiating healthy and pathological tissues within the bladder and potentially other hollow organs.

The spectrum registered by a reflected-light bright-field spectroscopic microscope (SM) can quantify the microscopically indiscernible, deeply subdiffractional length scales within samples such as biological cells and tissues. Nevertheless, quantification of biological specimens via any optical measures most often reveals ambiguous information about the specific structural properties within the studied samples. Thus, optical quantification remains nonintuitive to users from the diverse fields of technique application. In this work, we demonstrate that the SM signal can be analyzed to reconstruct explicit physical measures of internal structure within label-free, weakly scattering samples: characteristic length scale and the amplitude of spatial refractive-index (RI) fluctuations. We present and validate the reconstruction algorithm via finite-difference time-domain solutions of Maxwell’s equations on an example of exponential spatial correlation of RI. We apply the validated algorithm to experimentally measure structural properties within isolated cells from two genetic variants of HT29 colon cancer cell line as well as within a prostate tissue biopsy section. The presented methodology can lead to the development of novel biophotonics techniques that create two-dimensional maps of explicit structural properties within biomaterials: the characteristic size of macromolecular complexes and the variance of local mass density.

Simvastatin is one of the most frequently prescribed statins because of its efficacy in the treatment of hypercholesterolemia, reducing cardiovascular risk and related mortality. Determination of its side effects on different tissues is mandatory to improve safe use of this drug. In the present study, the effects of simvastatin on molecular composition and structure of healthy rat livers were investigated by Fourier transform infrared and Raman imaging. Simvastatin-treated groups received 50  mg/kg/day simvastatin for 30 days. The ratio of the area and/or intensity of the bands assigned to lipids, proteins, and nucleic acids were calculated to get information about the drug-induced changes in tissues. Loss of unsaturation, accumulation of end products of lipid peroxidation, and alterations in lipid-to-protein ratio were observed in the treated group. Protein secondary structure studies revealed significant decrease in α-helix and increase in random coil, while native β-sheet decreases and aggregated β-sheet increases in treated group implying simvastatin-induced protein denaturation. Moreover, groups were successfully discriminated using principal component analysis. Consequently, high-dose simvastatin treatment induces hepatic lipid peroxidation and changes in molecular content and protein secondary structure, implying the risk of liver disorders in drug therapy.

Previous studies suggest that altered corneal temperature may be a feature of schizophrenia, but the association between major depressive disorder (MDD) and corneal temperature has yet to be assessed. The aim of this study is to investigate whether eye temperature is different among MDD patients than among healthy individuals. We used a thermographic camera to measure and compare the temperature profile across the corneas of 16 patients with MDD and 16 age- and sex-matched healthy subjects. We found that the average corneal temperature between the two groups did not differ statistically, although clinical severity correlated positively with right corneal temperature. Corneal temperature may be an indicator of clinical severity in psychiatric disorders, including depression.

Optical coherence tomography (OCT) has been widely used to study mammalian embryonic development with the advantages of high spatial and temporal resolutions and without the need for any contrast enhancement probes. However, the limited imaging depth of traditional OCT might prohibit visualization of the full embryonic body. To overcome this limitation, we have developed a new methodology to enhance the imaging range of OCT in embryonic day (E) 9.5 and 10.5 mouse embryos using rotational imaging. Rotational imaging OCT (RI-OCT) enables full-body imaging of mouse embryos by performing multiangle imaging. A series of postprocessing procedures was performed on each cross-section image, resulting in the final composited image. The results demonstrate that RI-OCT is able to improve the visualization of internal mouse embryo structures as compared to conventional OCT.

Although structural changes on the sarcomere level of skeletal muscle are known to occur due to various pathologies, rigorous studies of the reduced sarcomere quality remain scarce. This can possibly be explained by the lack of an objective tool for analyzing and comparing sarcomere images across biological conditions. Recent developments in second harmonic generation (SHG) microscopy and increasing insight into the interpretation of sarcomere SHG intensity profiles have made SHG microscopy a valuable tool to study microstructural properties of sarcomeres. Typically, sarcomere integrity is analyzed by fitting a set of manually selected, one-dimensional SHG intensity profiles with a supramolecular SHG model. To circumvent this tedious manual selection step, we developed a fully automated image analysis procedure to map the sarcomere disorder for the entire image at once. The algorithm relies on a single-frequency wavelet-based Gabor approach and includes a newly developed normalization procedure allowing for unambiguous data interpretation. The method was validated by showing the correlation between the sarcomere disorder, quantified by the M-band size obtained from manually selected profiles, and the normalized Gabor value ranging from 0 to 1 for decreasing disorder. Finally, to elucidate the applicability of our newly developed protocol, Gabor analysis was used to study the effect of experimental autoimmune encephalomyelitis on the sarcomere regularity. We believe that the technique developed in this work holds great promise for high-throughput, unbiased, and automated image analysis to study sarcomere integrity by SHG microscopy.

Flow imaging is an important technique in a range of disease areas, but estimating low flow speeds, especially near the walls of blood vessels, remains challenging. Pulsed photoacoustic flow imaging can be an alternative since there is little signal contamination from background tissue with photoacoustic imaging. We propose flow imaging using a clinical photoacoustic system that is both handheld and portable. The system integrates a linear array with 7.5 MHz central frequency in combination with a high-repetition-rate diode laser to allow high-speed photoacoustic imaging—ideal for this application. This work shows the flow imaging performance of the system in vitro using microparticles. Both two-dimensional (2-D) flow images and quantitative flow velocities from 12 to 75  mm/s were obtained. In a transparent bulk medium, flow estimation showed standard errors of ∼7% the estimated speed; in the presence of tissue-realistic optical scattering, the error increased to 40% due to limited signal-to-noise ratio. In the future, photoacoustic flow imaging can potentially be performed in vivo using fluorophore-filled vesicles or with an improved setup on whole blood.

Full-field optical coherence tomography (FF-OCT) is a powerful tool for nondestructive assessment of biological tissue, i.e., for the structural examination of tissue in depth at a cellular resolution. Mostly known as a microscopy device for ex vivo analysis, FF-OCT has also been adapted to endoscopy setups since it shows good potential for in situ cancer diagnosis and biopsy guidance. Nevertheless, all the attempts to perform endoscopic FF-OCT imaging did not go beyond lab setups. We describe here, to the best of our knowledge, the first handheld FF-OCT endoscope based on a tandem interferometry assembly using incoherent illumination. A common-path passive imaging interferometer at the tip of an optical probe makes it robust and insensitive to environmental perturbations, and a low finesse Fabry–Perot processing interferometer guarantees a compact system. A good resolution (2.7  μm transverse and 6  μm axial) is maintained through the long distance, small diameter relay optics of the probe, and a good signal-to-noise ratio is achieved in a limited 100 ms acquisition time. High-resolution images and a movie of a rat brain slice have been recorded by moving the contact endoscope over the surface of the sample, allowing for tissue microscopic exploration at 20  μm under the surface. These promising ex vivo results open new perspectives for in vivo imaging of biological tissue, in particular, in the field of cancer and surgical margin assessment.

Cocaine abuse can lead to cerebral strokes and hemorrhages secondary to cocaine’s cerebrovascular effects, which are poorly understood. We assessed cocaine’s effects on cerebrovascular anatomy and function in the somatosensory cortex of the rat’s brain. Optical coherence tomography was used for in vivo imaging of three-dimensional cerebral blood flow (CBF) networks and to quantify CBF velocities (CBFv), and multiwavelength laser-speckle-imaging was used to simultaneously measure changes in CBFv, oxygenated (Δ[HbO₂]) and deoxygenated hemoglobin (Δ[HbR]) concentrations prior to and after an acute cocaine challenge in chronically cocaine exposed rats. Immunofluorescence techniques on brain slices were used to quantify microvasculature density and levels of vascular endothelial growth factor (VEGF). After chronic cocaine (2 and 4 weeks), CBFv in small vessels decreased, whereas vasculature density and VEGF levels increased. Acute cocaine further reduced CBFv and decreased Δ[HbO₂] and this decline was larger and longer lasting in 4 weeks than 2 weeks cocaine-exposed rats, which indicates that risk for ischemia is heightened during intoxication and that it increases with chronic exposures. These results provide evidence of cocaine-induced angiogenesis in cortex. The CBF reduction after chronic cocaine exposure, despite the increases in vessel density, indicate that angiogenesis was insufficient to compensate for cocaine-induced disruption of cerebrovascular function.

High-resolution optical coherence tomography (OCT) retinal imaging is important to noninvasively visualize the various retinal structures to aid in better understanding of the pathogenesis of vision-robbing diseases. However, conventional OCT systems have a trade-off between lateral resolution and depth-of-focus. In this report, we present the development of a focus-stacking OCT system with automatic focus optimization for high-resolution, extended-focal-range clinical retinal imaging by incorporating a variable-focus liquid lens into the sample arm optics. Retinal layer tracking and selection was performed using a graphics processing unit accelerated processing platform for focus optimization, providing real-time layer-specific en face visualization. After optimization, multiple volumes focused at different depths were acquired, registered, and stitched together to yield a single, high-resolution focus-stacked dataset. Using this system, we show high-resolution images of the retina and optic nerve head, from which we extracted clinically relevant parameters such as the nerve fiber layer thickness and lamina cribrosa microarchitecture.

We propose to use sparsely sampled line scans with a sparsity-based reconstruction method to obtain images in a wide field of view (WFOV) multifocal scanning microscope. In the WFOV microscope, we used a holographically generated irregular focus grid to scan the sample in one dimension and then reconstructed the sample image from line scans by measuring the transmission of the foci through the sample during scanning. The line scans were randomly spaced with average spacing larger than the Nyquist sampling requirement, and the image was recovered with sparsity-based reconstruction techniques. With this scheme, the acquisition data can be significantly reduced and the restriction for equally spaced foci positions can be removed, indicating simpler experimental requirement. We built a prototype system and demonstrated the effectiveness of the reconstruction by recovering microscopic images of a U.S. Air Force target and an onion skin cell microscope slide with 40, 60, and 80% missing data with respect to the Nyquist sampling requirement.

Fluorescent proteins and dyes are routine tools for biological research to describe the behavior of genes, proteins, and cells, as well as more complex physiological dynamics such as vessel permeability and pharmacokinetics. The use of these probes in whole body in vivo imaging would allow extending the range and scope of current biomedical applications and would be of great interest. In order to comply with a wide variety of application demands, in vivo imaging platform requirements span from wide spectral coverage to precise quantification capabilities. Fluorescence molecular tomography (FMT) detects and reconstructs in three dimensions the distribution of a fluorophore in vivo. Noncontact FMT allows fast scanning of an excitation source and noninvasive measurement of emitted fluorescent light using a virtual array detector operating in free space. Here, a rigorous process is defined that fully characterizes the performance of a custom-built horizontal noncontact FMT setup. Dynamic range, sensitivity, and quantitative accuracy across the visible spectrum were evaluated using fluorophores with emissions between 520 and 660 nm. These results demonstrate that high-performance quantitative three-dimensional visible light FMT allowed the detection of challenging mesenteric lymph nodes in vivo and the comparison of spectrally distinct fluorescent reporters in cell culture.

The Fourier ptychography technique in reflection mode has great potential applications in tissue imaging and optical inspection, but the current configuration either has a limitation on cut-off frequency or is not practical. By placing the imaging aperture stop outside the illumination path, the illumination numerical aperture (NA) can be greater than the imaging NA of the objective lens. Thus, the cut-off frequency achieved in the proposed optical system is greater than twice the objective’s NA divided by the wavelength (2NA_obj/λ), which is the diffraction limit for the cut-off frequency in an incoherent epi-illumination configuration. We experimentally demonstrated that the synthesized NA is increased by a factor of 4.5 using the proposed optical concept. The key advantage of the proposed system is that it can achieve high-resolution imaging over a large field of view with a simple objective. It will have a great potential for applications in endoscopy, biomedical imaging, surface metrology, and industrial inspection.

Laser speckle imaging (LSI) is an interferometric technique that provides information about the relative speed of moving scatterers in a sample. Photothermal LSI overcomes limitations in depth resolution faced by conventional LSI by incorporating an excitation pulse to target absorption by hemoglobin within the vascular network. Here we present results from experiments designed to determine the mechanism by which photothermal LSI decreases speckle contrast. We measured the impact of mechanical properties on speckle contrast, as well as the spatiotemporal temperature dynamics and bulk convective motion occurring during photothermal LSI. Our collective data strongly support the hypothesis that photothermal LSI achieves a transient reduction in speckle contrast due to bulk motion associated with thermally driven convection. The ability of photothermal LSI to image structures below a scattering medium may have important preclinical and clinical applications.

Fluorescence molecular tomography (FMT) is a rapidly growing imaging method that facilitates the recovery of small fluorescent targets within biological tissue. The major challenge facing the FMT reconstruction method is the ill-posed nature of the inverse problem. In order to overcome this problem, the acquisition of large FMT datasets and the utilization of a fast FMT reconstruction algorithm with sparsity regularization have been suggested recently. Therefore, the use of a joint L1/total-variation (TV) regularization as a means of solving the ill-posed FMT inverse problem is proposed. A comparative quantified analysis of regularization methods based on L1-norm and TV are performed using simulated datasets, and the results show that the fast composite splitting algorithm regularization method can ensure the accuracy and robustness of the FMT reconstruction. The feasibility of the proposed method is evaluated in an in vivo scenario for the subcutaneous implantation of a fluorescent-dye-filled capillary tube in a mouse, and also using hybrid FMT and x-ray computed tomography data. The results show that the proposed regularization overcomes the difficulties created by the ill-posed inverse problem.

A combined diffuse speckle contrast analysis (DSCA)–near-infrared spectroscopy (NIRS) system is proposed to simultaneously measure qualitative blood flow and blood oxygenation changes in human tissue. The system employs an optical switch to alternate two laser sources at two different wavelengths and a CCD camera to capture the speckle image. Therefore, an optical density can be measured from two wavelengths for NIRS measurements and a speckle contrast can be calculated for DSCA measurements. In order to validate the system, a flow phantom test and an arm occlusion protocol for arterial and venous occlusion were performed. Shorter exposure times (<1  ms) show a higher drop (between 50% and 66%) and recovery of 1/K_S² values after occlusion (approximately 150%), but longer exposure time (3 ms) shows more consistent hemodynamic changes. For four subjects, the 1/K_S² values dropped to an average of 82.1±4.0% during the occlusion period and the average recovery of 1/K_S² values after occlusion was 109.1±0.8%. There was also an approximately equivalent amplitude change in oxyhemoglobin (OHb) and deoxyhemoglobin (RHb) during arterial occlusion (max RHb=0.0085±0.0024  mM/DPF, min OHb=−0.0057±0.0044  mM/DPF). The sensitivity of the system makes it a suitable modality to observe qualitative hemodynamic trends during induced physiological changes.

Spasticity is a motor disorder frequently present in individuals with cerebral palsy (CP). This study aimed to evaluate the effect of low-level laser therapy (LLLT) on the spasticity of the masseter and anterior temporal muscle fibers in children with CP over three weeks of intermittent laser exposures. The bite force (BF) of the masticatory muscles and the amplitude of mouth opening were evaluated before and after laser irradiation in 30 children with CP. Both sides of the masseter and temporalis muscles were irradiated with low-intensity diode laser pulses of 808-nm wavelength six times over three consecutive weeks. During the subsequent three weeks of postlaser exposures, although no laser treatment was applied, the evaluation parameters were measured and recorded. A significant improvement in the amplitude of mouth opening and a decrease in the BF were observed in the weeks following LLLT (P<0.05). However, by the sixth week post-LLLT, the BF and the amplitude of mouth opening reverted to values equivalent to those obtained before the first application of LLLT. Our investigation revealed low-level energy exposures from a 808-nm diode laser to be an effective short-term therapeutic tool. This method increased the amplitude of mouth opening and decreased the muscle tonus of children with spastic CP over a time course of three weeks of intermittent laser applications.

Online light dosimetry with real-time feedback was applied for temoporfin-mediated interstitial photodynamic therapy (PDT) of dog prostate. The aim was to investigate the performance of online dosimetry by studying the correlation between light dose plans and the tissue response, i.e., extent of induced tissue necrosis and damage to surrounding organs at risk. Light-dose planning software provided dose plans, including light source positions and light doses, based on ultrasound images. A laser instrument provided therapeutic light and dosimetric measurements. The procedure was designed to closely emulate the procedure for whole-prostate PDT in humans with prostate cancer. Nine healthy dogs were subjected to the procedure according to a light-dose escalation plan. About 0.15  mg/kg temoporfin was administered 72 h before the procedure. The results of the procedure were assessed by magnetic resonance imaging, and gross pathology and histopathology of excised tissue. Light dose planning and online dosimetry clearly resulted in more focused effect and less damage to surrounding tissue than interstitial PDT without dosimetry. A light energy dose–response relationship was established where the threshold dose to induce prostate gland necrosis was estimated from 20 to 30  J/cm².

This PDF file contains the errata for “JBO Vol. 21 Issue 02 Paper JBO-2016-0127-ERR” for JBO Vol. 21 Issue 02

This PDF file contains the errata for “JBO Vol. 21 Issue 02 Paper JBO-2016-0215-ERR” for JBO Vol. 21 Issue 02