High-resolution optical longitudinal cortical imaging usually uses cranial window, which involves removing a skull portion and sealing the exposed brain area with a transparent cover glass, allowing ballistic photons to reach the cortex with minimal disturbance of the brain function. It enables obtaining high-resolution brain images in extended periods of time for long-term neuronal activity studies using confocal and two-photon microscopies. Photoacoustic microscopy (PAM), as the only imaging method that directly measure absorption contrast, is a complementary functional imaging method to provide absorption related brain information, such as total concentration of hemoglobin and oxygen saturation of hemoglobin. However, the use of traditional piezoelectric transducers (PZT) to collect ultrasound signal greatly limits the versatility of PAM. Though highly sensitive, PZT transducers are usually bulky and optically opaque. It blocks the light and is hard to be inserted into the limited distance between the optical objective and imaging sample, which are normally less than one millimeter when using a high- numerical aperture (NA) objective to achieve submicron resolution.
Here, we developed a simple and cost-efficient soft nanoimprint lithography (NIL) process to fabricate fully embedded micro-ring resonator ultrasound detectors on optically transparent substrates, and integrated the detector onto a cranial window, making cranial window itself an ultrasonic detector. We implanted this functional cranial window on mouse head and achieved longitudinal monitoring of cortex vasculature using PAM. Our low-cost, disposable, and optically transparent detector may potentially reshape the longitudinal functional brain imaging using PAM in small animals.
Visible-light optical coherence tomography (vis-OCT) is an emerging imaging modality, providing new capabilities in both anatomical and functional imaging of biological tissue. It relies on visible light illumination, whereas most commercial and investigational OCTs use near-infrared light. As a result, vis-OCT requires different considerations in engineering design and implementation but brings unique potential benefits to both fundamental research and clinical care of several diseases. Here, we intend to provide a summary of the development of vis-OCT and its demonstrated applications. We also provide perspectives on future technology improvement and applications.
Retinal oxygen metabolic rate can be effectively measured by visible-light optical coherence tomography (vis-OCT), which simultaneously quantifies oxygen saturation and blood flow rate in retinal vessels through spectroscopic analysis and Doppler measurement, respectively. Doppler OCT relates phase variation between sequential A-lines to the axial flow velocity of the scattering medium. The detectable phase shift is between −π and π due to its periodicity, which limits the maximum measurable unambiguous velocity without phase unwrapping. Using shorter wavelengths, vis-OCT is more vulnerable to phase ambiguity since flow induced phase variation is linearly related to the center wavenumber of the probing light. We eliminated the need for phase unwrapping using spectroscopic Doppler analysis. We split the whole vis-OCT spectrum into a series of narrow subbands and reconstructed vis-OCT images to extract corresponding Doppler phase shifts in all the subbands. Then, we quantified flow velocity by analyzing subband-dependent phase shift using linear regression. In the phantom experiment, we showed that spectroscopic Doppler analysis extended the measurable absolute phase shift range without conducting phase unwrapping. We also tested this method to quantify retinal blood flow in rodents in vivo.
KEYWORDS: Optical coherence tomography, Optical imaging, 3D scanning, Injuries, 3D modeling, Animal model studies, 3D image processing, Laser scanners, Biopsy
High-resolution colposcopic optical coherence tomography (OCT) provides key anatomical measures, such as thickness and minor traumatic injury of vaginal epithelium, of the female reproductive tract noninvasively. This information can be helpful in both fundamental investigations in animal models and disease screenings in humans. We present a fiber-based visible-light OCT and two probe designs for colposcopic application. One probe conducts circular scanning using a DC motor, and the other probe is capable of three-dimensional imaging over a 4.6×4.6-mm2 area using a pair of galvo scanners. Using this colposcopic vis-OCT with both probes, we acquired high-resolution images from whole isolated macaque vaginal samples and identified biopsy lesions.
Monitoring cortical hemodynamic response after ischemic stroke (IS) is essential for understanding the pathophysiological mechanisms behind IS-induced neuron loss. Functional optical coherence tomography (OCT) is an emerging technology that can fulfill the requirement, providing label-free, high-resolution 3D images of cerebral hemodynamics.
Unfortunately, strong tissue scattering pose a significant challenge for existing OCT oximetry techniques, as they either ignore the effect or compensate it numerically. Here we developed a novel dual-depth sampling and normalization strategy using visible-light OCT (vis-OCT) angiograms that can provide robust and precise sO2 estimations within cerebral circulation. The related theoretical formulation were established, and its implication and limitations were discussed.
We monitored mouse cortical hemodynamics using the newly-developed method. Focal ischemic stroke was induced through photothrombosis. The analysis on pre- and post-IS vis-OCT images revealed both vascular morphology and oxygenation altered substantially after the occlusion. First, the ischemic core could be clearly identified as angiographic intensity fell below the detection limit. In addition, vessel dilation presented universally in the penumbra region. Notably for pial arteriles, the percentage of increase demonstrated inverse relationship with their pre-occlusion, pre-dilation dimeter.
Vis-OCT oxygenation maps on intact cortex revealed spatial sO2 variations within pial vessels. Specifically, sO2 in arterioles decreased as it bifurcated and plunged into deeper tissue. Similarly, venous sO2 was higher in the larger, more superficial pial brunches. However, such difference was no longer appreciable after photothrombosis. Averaged arteriole sO2 dropped to 64% – 67% in the penumbra region.
We developed a simultaneous visible-light (Vis) and near-infrared (NIR) dual-band optical coherence tomography (OCT) system using a single supercontinuum laser source. The goal was to benchmark our newly developed Vis-OCT against the well-developed NIR-OCT. The Vis-OCT subsystem operated at 91 nm full-width-at-half-maximum (FWHM) bandwidth centered at 566 nm; the NIR-OCT subsystem operated at 93 nm FWHM bandwidth centered at 841 nm. The axial resolutions were 1.8 and 4.4 μm in air for the Vis- and NIR-OCT subsystems, respectively. We compared the respective performances, including anatomical imaging, angiography, absolute retinal blood flow measurements, and spectroscopic analysis for retinal blood oxygen saturation (sO2), between the two subsystems in rodents in vivo. While demonstrating minor discrepancies related to operation wavelengths, both subsystems showed comparable performances in the first three tests. However, we were only able to retrieve sO2 using the Vis-OCT subsystem.
The melanin in the retinal pigment epithelium (RPE) protects retina and other ocular tissues by photo-screening and acting as antioxidant and free radical scavenger. It helps maintain normal visual functions since human eye is subjected to lifelong high oxygen stress and photon exposure. Loss of the RPE melanin weakens the protection mechanism and jeopardizes ocular health. Local decrease in the RPE melanin concentration is believed to be both a cause and a sign of early-stage age-related macular degeneration (AMD), the leading blinding disease in developed world. Current technology cannot quantitatively measure the RPE melanin concentration which might be a promising marker in early AMD screening. Photoacoustic ophthalmoscopy (PAOM), as an emerging optical absorption-based imaging technology, can potentially be applied to measure the RPE melanin concentration if the dependence of the detectable photoacoustic (PA) signal amplitudes on the RPE melanin concentrations is verified. In this study, we tested the feasibility of using PA signal ratio from RPE melanin and the nearby retinal blood vessels as an indicator of the RPE melanin variation. A novel whole eye optical model was designed and Monte Carlo modeling of light (MCML) was employed. We examined the influences on quantification from PAOM axial resolution, the depth and diameter of the retinal blood vessel, and the RPE thickness. The results show that the scheme is robust to individual histological and illumination variations. This study suggests that PAOM is capable of quantitatively measuring the RPE melanin concentration in vivo.
The retinal pigment epithelium (RPE) melanin plays an important role in maintaining normal visual functions. A decrease in the RPE melanin concentration with aging is believed to be associated with several blinding diseases, including age-related macular degeneration. Quantifying the RPE melanin noninvasively is therefore important in evaluating the retinal health and aging conditions. Photoacoustic ophthalmoscopy (PAOM), as an optical absorption-based imaging technology, can potentially be applied to measure variations in the RPE melanin if the relationship between the detected photoacoustic (PA) signal amplitudes and the RPE melanin concentrations can be established. In this work, we tested the feasibility of using PA signals from retinal blood vessels as references to measure RPE melanin variation using Monte Carlo (MC) simulation. The influences from PAOM axial resolution, the depth and diameter of the retinal blood vessel, and the RPE thickness were examined. We proposed a calibration scheme by relating detected PA signals to the RPE melanin concentrations, and we found that the scheme is robust to these tested parameters. This study suggests that PAOM has the capability of quantitatively measuring the RPE melanin in vivo.
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