Deep ultraviolet fluorescence microscopy of three-dimensional structures in the mouse brain.

Three-dimensional (3D) imaging at cellular resolution improves our understanding of the brain architecture and is crucial for structural and functional integration as well as for the understanding of normal and pathological conditions in the brain. We developed a wide-field fluorescent microscope for 3D imaging of the brain structures using deep ultraviolet (DUV) light. This microscope allowed fluorescence imaging with optical sectioning due to the large absorption at the surface of the tissue and hence low tissue penetration of DUV light. Multiple channels of fluorophore signals were detected using single or a combination of dyes emitting fluorescence in the visible range of spectrum upon DUV excitation. Combination of this DUV microscope with microcontroller-based motorized stage enabled wide-field imaging of a coronal section of the cerebral hemisphere in mouse for deciphering cytoarchitecture of each substructure in detail. We extended this by integrating vibrating microtome which allowed serial block-face imaging of the brain structure such as the habenula in mouse. Acquired images were with resolution high enough for quantification of the cell numbers and density in the mouse habenula. Upon block-face imaging of the tissues covering entire extent of the cerebral hemisphere of the mouse brain, acquired data were registered and segmented for quantification of cell number in each brain regions. Results in the current analysis indicated that this novel microscope could be a convenient tool for large-scale 3D analysis of the brain in mice.

A Novel Microcontroller-Based System for the Wheel-Running Activity in Mice.

Voluntary wheel-running activity is a way to assess rodents' circadian rhythm and motivation for exercise. Deficits in these behaviors are implicated in the pathophysiology of sleep and psychiatric disorders. Limited space in animal facilities can hamper long-term monitoring of running wheel activity outside of the home cage. To address this issue, we provide a stand-alone solution to monitor the wheel-running activity of mice in their home cage. This system, named the wheel-running activity acquisition (WRAQ) system, is based on a microcontroller driven by a lithium polymer battery. With the WRAQ, we can record the wheel-running activity and illumination data for at least 30 d. Applying the WRAQ to an endotoxemia mouse model robustly detected the altered wheel-running activity and its recovery. With wireless data transfer capability extension, the system also allows for online monitoring and reporting of the circadian time (CT). We used the online monitoring of wheel-running activity with this extended WRAQ system and observed a significant shift of the active period in the circadian rhythm following a temporal chemogenetic activation of the suprachiasmatic nucleus (SCN)-subparaventricular zone (SPZ). Together, these findings indicate that the WRAQ system is a novel and cost-effective solution for the analysis of wheel-running activity in mice.

Clinical multi-functional OCT for retinal imaging.

A compact clinical prototype multi-functional optical coherence tomography (OCT) device for the posterior human eye has been developed. This compact Jones-matrix OCT (JM-OCT) device integrates all components into a single package. Multiple image functions, i.e., scattering intensity, OCT angiography, and the degree of polarization uniformity, are obtained. The device has the capability for measuring local birefringence. Multi-functional imaging of several eyes with age-related macular degeneration is demonstrated. The compact JM-OCT device will be useful for the in vivo non-invasive investigation of abnormal tissues.

Machine-learning based segmentation of the optic nerve head using multi-contrast Jones matrix optical coherence tomography with semi-automatic training dataset generation.

A pixel-by-pixel tissue classification framework using multiple contrasts obtained by Jones matrix optical coherence tomography (JM-OCT) is demonstrated. The JM-OCT is an extension of OCT that provides OCT, OCT angiography, birefringence tomography, degree-of-polarization uniformity tomography, and attenuation coefficient tomography, simultaneously. The classification framework consists of feature engineering, k-means clustering that generates a training dataset, training of a tissue classifier using the generated training dataset, and tissue classification by the trained classifier. The feature engineering process generates synthetic features from the primary optical contrasts obtained by JM-OCT. The tissue classification is performed in the feature space of the engineered features. We applied this framework to the in vivo analysis of optic nerve heads of posterior eyes. This classified each JM-OCT pixel into prelamina, lamina cribrosa (lamina beam), and retrolamina tissues. The lamina beam segmentation results were further utilized for birefringence and attenuation coefficient analysis of lamina beam.

Comparison of intensity, phase retardation, and local birefringence images for filtering blebs using polarization-sensitive optical coherence tomography.

Polarization-sensitive optical coherence tomography (PS-OCT) allows the recording of depth-resolved polarimetric measurements. It has been reported that phase retardation and local birefringence images can noninvasively detect fibrotic area in blebs after glaucoma surgery. Evaluation of scar fibrosis in blebs is important not only for predicting bleb function, but also for planning revision trabeculectomy. Herein, we characterize the intensity, phase retardation, and local birefringence images of blebs using PS-OCT. A total of 85 blebs from 85 patients who had undergone trabeculectomy were examined. Both phase retardation and local birefringence images detected fibrotic changes in blebs after glaucoma surgery. Phase retardation images detected slight fibrotic change during the early stage after surgery, whereas local birefringence images showed localized fibrotic tissue. There are two main patterns of local birefringence image changes in blebs: plate-like birefringence changes and diffuse changes. The area of plate-like birefringence change was significantly larger in poorly functioning blebs and is thus correlated with bleb function. These data suggest that the plate-like fibrotic change evaluation by PS-OCT may be useful not only for noninvasive evaluation of fibrotic scar tissue in blebs, but also for developing strategies for revision trabeculectomy.

Generation and optimization of superpixels as image processing kernels for Jones matrix optical coherence tomography.

Jones matrix-based polarization sensitive optical coherence tomography (JM-OCT) simultaneously measures optical intensity, birefringence, degree of polarization uniformity, and OCT angiography. The statistics of the optical features in a local region, such as the local mean of the OCT intensity, are frequently used for image processing and the quantitative analysis of JM-OCT. Conventionally, local statistics have been computed with fixed-size rectangular kernels. However, this results in a trade-off between image sharpness and statistical accuracy. We introduce a superpixel method to JM-OCT for generating the flexible kernels of local statistics. A superpixel is a cluster of image pixels that is formed by the pixels' spatial and signal value proximities. An algorithm for superpixel generation specialized for JM-OCT and its optimization methods are presented in this paper. The spatial proximity is in two-dimensional cross-sectional space and the signal values are the four optical features. Hence, the superpixel method is a six-dimensional clustering technique for JM-OCT pixels. The performance of the JM-OCT superpixels and its optimization methods are evaluated in detail using JM-OCT datasets of posterior eyes. The superpixels were found to well preserve tissue structures, such as layer structures, sclera, vessels, and retinal pigment epithelium. And hence, they are more suitable for local statistics kernels than conventional uniform rectangular kernels.

Three-dimensional multi-contrast imaging of in vivo human skin by Jones matrix optical coherence tomography.

A custom made dermatological Jones matrix optical coherence tomography (JM-OCT) is presented. It uses a passive-polarization-delay component based swept-source JM-OCT configuration, but is specially designed for in vivo human skin measurement. The center wavelength of its probe beam is 1310 nm and the A-line rate is 49.6 kHz. The JM-OCT is capable of simultaneously providing birefringence (local retardation) tomography, degree-of-polarization-uniformity tomography, complex-correlation-based optical coherence angiography, and conventional scattering OCT. To evaluate the performance of this JM-OCT, we measured in vivo human skin at several locations. Using the four kinds of OCT contrasts, the morphological characteristics and optical properties of different skin types were visualized.

Noise stochastic corrected maximum a posteriori estimator for birefringence imaging using polarization-sensitive optical coherence tomography.

This paper presents a noise-stochastic corrected maximum a posteriori estimator for birefringence imaging using Jones matrix optical coherence tomography. The estimator described in this paper is based on the relationship between probability distribution functions of the measured birefringence and the effective signal to noise ratio (ESNR) as well as the true birefringence and the true ESNR. The Monte Carlo method is used to numerically describe this relationship and adaptive 2D kernel density estimation provides the likelihood for a posteriori estimation of the true birefringence. Improved estimation is shown for the new estimator with stochastic model of ESNR in comparison to the old estimator, both based on the Jones matrix noise model. A comparison with the mean estimator is also done. Numerical simulation validates the superiority of the new estimator. The superior performance of the new estimator was also shown by in vivo measurement of optic nerve head.

Quantitative Evaluation of Phase Retardation in Filtering Blebs Using Polarization-Sensitive Optical Coherence Tomography.

PURPOSE: Polarization-sensitive optical coherence tomography (PS-OCT) can detect and evaluate scar fibrosis of the filtering blebs after glaucoma surgery. Although the change in phase retardation reportedly reflects bleb function, quantitative assessment of phase retardation in ocular tissues has not been conducted. We aimed to establish quantitative methods to investigate changes in phase retardation in the blebs after surgery using PS-OCT. METHODS: Twenty-two blebs of 22 patients who had undergone glaucoma filtration surgery were consecutively examined for 4 months. Phase retardation was measured by PS-OCT and quantitatively analyzed to evaluate its relationship with bleb function based on intraocular pressure and medication use. Cross-sectional re-evaluation was also performed for a previous data set of 153 blebs of 122 patients. RESULTS: In consecutive measurements, all blebs showed a low phase retardation value and good bleb function until 2 weeks. One month postoperatively, the phase retardation value was significantly increased, whereas bleb function was still good. The phase retardation value at 1 month postoperatively was significantly correlated with bleb function at 4 months postoperatively. While 55.6% of blebs with a high phase retardation value at 1 month subsequently lost function, only 7.7% with a low phase retardation value had bleb failure. In the cross-sectional re-evaluation, the quantitatively evaluated phase retardation value was highly correlated with bleb function (ß = 0.770, P < 0.001). CONCLUSIONS: An increase in phase retardation preceded deterioration of bleb function. The change in phase retardation may provide a prognostic metric for bleb function in the early stage after surgery.

Objective Evaluation of Functionality of Filtering Bleb Based on Polarization-Sensitive Optical Coherence Tomography.

PURPOSE: The fibrosis score is a new diagnostic score that we have developed to evaluate the function of bleb structures after glaucoma filtration surgery using polarization-sensitive optical coherence tomography (PS-OCT). This study aims to assess the efficacy of the fibrosis score in discriminating nonfunctional from the functional blebs. METHODS: A total of 20 patients who had undergone glaucoma filtration surgery were imaged at different time periods after surgery using PS-OCT. Birefringence tomography of blebs was obtained from PS-OCT, and the fibrosis score was computed for each patient. The fibrosis score is defined as the area of occupation of high birefringence area in the conjunctiva. The blebs were classified as functional or nonfunctional according to the IOP and the application of medication. The power of the fibrosis score to discriminate nonfunctional blebs from functional blebs was evaluated. RESULTS: The difference in the mean fibrosis score between the functional and nonfunctional bleb group was statistically significant. The fibrosis score showed good ability to discriminate nonfunctional from functional blebs. The area under the receiver operating characteristic curve was 0.82. The best combination of the sensitivity and specificity was 67% and 100%, respectively, for classifying nonfunctional cases. CONCLUSIONS: The fibrosis score showed a high ability to discriminate nonfunctional from functional blebs. Polarization-sensitive OCT is a noninvasive technique that provides not only the fibrosis score but also standard structural tomography. It can be a comprehensive tool for longitudinal evaluation after filtration surgery for glaucoma.

Birefringence imaging of posterior eye by multi-functional Jones matrix optical coherence tomography.

A clinical grade prototype of posterior multifunctional Jones matrix optical coherence tomography (JM-OCT) is presented. This JM-OCT visualized depth-localized birefringence in addition to conventional cumulative phase retardation imaging through local Jones matrix analysis. In addition, it simultaneously provides a sensitivity enhanced scattering OCT, a quantitative polarization uniformity contrast, and OCT-based angiography. The probe beam is at 1-µm wavelength band. The measurement speed and the depth-resolution were 100,000 A-lines/s, and 6.6 µm in tissue, respectively. Normal and pathologic eyes are examined and several clinical features are revealed, which includes high birefringence in the choroid and lamina cribrosa, and birefringent layered structure of the sclera. The theoretical details of the depth-localized birefringence imaging and conventional phase retardation imaging are formulated. This formulation indicates that the birefringence imaging correctly measures a depth-localized single-trip phase retardation of a tissue, while the conventional phase retardation can provide correct single-trip phase retardation only for some specific types of samples.

Repeatability of corneal phase retardation measurements by polarization-sensitive optical coherence tomography.

PURPOSE: Polarization-sensitive optical coherence tomography (PS-OCT) can evaluate internal tissue structures of the cornea, such as collagen fibers, by phase retardation measurement. In this study, we assessed the repeatability of corneal phase retardation measurements using anterior segment PS-OCT. METHODS: A total of 173 eyes of 173 patients were measured using PS-OCT. In total, 58 eyes of young subjects with normal corneas, 28 eyes of old subjects with normal corneas, 26 eyes with corneal dystrophy or degeneration, 37 eyes with corneal transplantation, and 24 eyes with keratoconus were evaluated. The 3-mm diameter average of en face phase retardation of the posterior corneal surface was examined using PS-OCT. To evaluate the repeatability, intraclass correlation coefficients (ICCs) were calculated for intraobserver repeatability and interobserver repeatability analysis. RESULTS: Polarization-sensitive OCT showed good repeatability for corneal measurements. Intraclass correlation coefficients of intraobserver and interobserver repeatability of all the subjects were 0.989 and 0.980, respectively. Intraclass correlation coefficients of the intraobserver for each group, that is, young and old subjects with normal cornea, cornea dystrophy/degeneration, corneal transplantation, and keratoconus, were 0.961, 0.975, 0.984, 0.978, and 0.996, respectively. Interobserver ICCs for the above-mentioned respective groups were 0.952, 0.964, 0.988, 0.959, and 0.975, respectively. CONCLUSIONS: Polarization-sensitive OCT showed good repeatability for phase retardation measurements of central corneas not only for normal corneas, but also for various diseased corneas. Polarization-sensitive OCT might be useful for evaluating corneal phase retardation, which is one of the parameters that defines birefringence.

Noninvasive evaluation of phase retardation in blebs after glaucoma surgery using anterior segment polarization-sensitive optical coherence tomography.

PURPOSE: Evaluation of bleb morphology using anterior segment optical coherence tomography (OCT) can offer important information regarding bleb function after glaucoma surgery. However, analysis of tissue properties, such as scar fibrosis of blebs, is difficult with conventional OCT. The birefringence of the blebs as susceptible measure of fibrosis scar was evaluated using polarization-sensitive OCT (PS-OCT) and its relation with bleb function was assessed. METHODS: One hundred and fifty-three blebs of 122 patients that had undergone trabeculectomy or an Ex-Press tube shunt were examined. Also, in 14 blebs of 12 patients, consecutive measurements were performed for 2 months after surgery. The birefringence of blebs was evaluated by measuring alteration of phase retardation using PS-OCT. Functionality of the bleb was classified according to IOP and medication. The bleb morphology in terms of size and characteristics was evaluated using three-dimensional (3D) cornea and anterior segment OCT. RESULTS: The alteration of phase retardation of blebs had the largest impact on bleb functionality than bleb morphology as shown by multiple regression analysis. In consecutive measurements, no blebs showed abnormal phase retardation until 1 week after surgery. Some blebs showed partial increase of phase retardation at 1 month after surgery. CONCLUSIONS: Intrableb fibrosis can be noninvasively evaluated with PS-OCT. Evaluation of birefringence by measuring phase retardation alterations using PS-OCT suggests new approaches for the postoperative management of glaucoma blebs regarding antifibrotic treatment for preventing IOP increases.

Bayesian maximum likelihood estimator of phase retardation for quantitative polarization-sensitive optical coherence tomography.

This paper presents the theory and numerical implementation of a maximum likelihood estimator for local phase retardation (i.e., birefringence) measured using Jones-matrix-based polarization sensitive optical coherence tomography. Previous studies have shown conventional mean estimations of phase retardation and birefringence are significantly biased in the presence of system noise. Our estimator design is based on a Bayes' rule that relates the distributions of the measured birefringence under a particular true birefringence and the true birefringence under a particular measured birefringence. We used a Monte-Carlo method to calculate the likelihood function that describes the relationship between the distributions and numerically implement the estimator. Our numerical and experimental results show that the proposed estimator was asymptotically unbiased even with low signal-to-noise ratio and/or for the true phase retardations close to the edge of the measurement range. The estimator revealed detailed clinical features when applied to the in vivo anterior human eye.

Conical scan polarization-sensitive optical coherence tomography.

We report on a new articular cartilage imaging technique with potential for clinical arthroscopic use, by supplementing the variable-incidence-angle polarization-sensitive optical coherence tomography method previously developed by us with a conical beam scan protocol. The technique is validated on bovine tendon by comparing experimental data with simulated data generated using the extended Jones matrix calculus. A unique capability of this new optical technique is that it can locate the "brushing direction" of collagen fibers in articular cartilage, which is structural information that extends beyond established methods such as split-line photography or birefringent fast-axis measurement in that it is uniquely defined over the full azimuthal-angle range of (-π, + π). The mapping of this direction over the cartilage surface may offer insights into the optimal design of tissue-engineering scaffolds for cartilage repair.

Polarization-sensitive optical coherence tomography using continuous polarization modulation with arbitrary phase modulation amplitude.

We demonstrate theoretically and experimentally that the phase retardance and relative optic-axis orientation of a sample can be calculated without prior knowledge of the actual value of the phase modulation amplitude when using a polarization-sensitive optical coherence tomography system based on continuous polarization modulation (CPM-PS-OCT). We also demonstrate that the sample Jones matrix can be calculated at any values of the phase modulation amplitude in a reasonable range depending on the system effective signal-to-noise ratio. This has fundamental importance for the development of clinical systems by simplifying the polarization modulator drive instrumentation and eliminating its calibration procedure. This was validated on measurements of a three-quarter waveplate and an equine tendon sample by a fiber-based swept-source CPM-PS-OCT system.

Experimental validation of an extended Jones matrix calculus model to study the 3D structural orientation of the collagen fibers in articular cartilage using polarization-sensitive optical coherence tomography.

We report results to verify a theoretical framework to analyze the 3D depth-wise structural organization of collagen fibers in articular cartilage using polarization-sensitive optical coherence tomography. Apparent birefringence data obtained from multi-angle measurements using a time domain polarization-sensitive optical coherence tomography system has been compared with simulated data based on the extended Jones matrix calculus. Experimental data has been shown to agree with the lamellar model previously proposed for the cartilage microstructure based on scanning electron microscopy data. This tool could have potential application in mapping the collagen structural orientation information of cartilage non-invasively during arthroscopy.

Comparative study of the angle-resolved backscattering properties of collagen fibers in bovine tendon and cartilage.

In a biological tissue, light scattering is based on the size and type of scatterers seen as refractive index variations that describe the optical properties shown. In this paper, we have implemented the variable incidence angle technique of multiple angle of illumination experiment on tendon and cartilage samples whose dominant constituents are genetically different types of collagen fibers, type I and type II, respectively. It is found that tendon displays a much greater angular anisotropy in its optical backscattering coefficient than the healthy cartilage. We propose that this is due to a more uniform distribution of fine fibrils than is found in tendon. Rayleigh-Gans approximation is used to give qualitative support to this idea.

Method to calibrate phase fluctuation in polarization-sensitive swept-source optical coherence tomography.

We present a phase fluctuation calibration method for polarization-sensitive swept-source optical coherence tomography (PS-SS-OCT) using continuous polarization modulation. The method uses a low-voltage broadband polarization modulator driven by a synchronized sinusoidal burst waveform rather than an asynchronous waveform, together with the removal of the global phases of the measured Jones matrices by the use of matrix normalization. This makes it possible to average the measured Jones matrices to remove the artifact due to the speckle noise of the signal in the sample without introducing auxiliary optical components into the sample arm. This method was validated on measurements of an equine tendon sample by the PS-SS-OCT system.

Performance comparison between 8- and 14-bit-depth imaging in polarization-sensitive swept-source optical coherence tomography.

Recently the effects of reduced bit-depth acquisition on swept-source optical coherence tomography (SS-OCT) image quality have been evaluated by using simulations and empirical studies, showing that image acquisition at 8-bit depth allows high system sensitivity with only a minimal drop in the signal-to-noise ratio compared to higher bit-depth systems. However, in these studies the 8-bit data is actually 12- or 14-bit ADC data numerically truncated to 8 bits. In practice, a native 8-bit ADC could actually possess a true bit resolution lower than this due to the electronic jitter in the converter etc. We compare true 8- and 14-bit-depth imaging of SS-OCT and polarization-sensitive SS-OCT (PS-SS-OCT) by using two hardware-synchronized high-speed data acquisition (DAQ) boards. The two DAQ boards read exactly the same imaging data for comparison. The measured system sensitivity at 8-bit depth is comparable to that for 14-bit acquisition when using the more sensitive of the available full analog input voltage ranges of the ADC. Ex-vivo structural and birefringence images of equine tendon indicate no significant differences between images acquired by the two DAQ boards suggesting that 8-bit DAQ boards can be employed to increase imaging speeds and reduce storage in clinical SS-OCT/PS-SS-OCT systems. One possible disadvantage is a reduced imaging dynamic range which can manifest itself as an increase in image artifacts due to strong Fresnel reflection.