Tissue metabolism is an inter-and intra-cellular process, which is essential to maintain the living state of tissue. Functional abnormality of such cellular processes initiates several metabolic diseases including non-alcoholic-fatty-liver-disease (NAFLD), liver fibrosis, cancer, chronic kidney disease, and so on. To understand the metabolic processes and how tissue metabolism changes from healthy to diseased conditions, it is required to investigate tissue metabolic activity in animal models in a label-free manner. Since the metabolic structures in animal organs extend into the deep region, deep tissue metabolism imaging is necessary. In this paper, we demonstrate the investigation of ex vivo animal organ models’ metabolism including mouse liver and kidney tissues using OCT-based dynamics imaging method. The dynamics imaging method is based on logarithmic intensity variance (LIV) analysis that enables volumetric metabolic imaging with a 50-kHz swept-source OCT system. NAFLD model mouse livers and obstructed mouse kidney model were investigated ex vivo. In the normal liver, highly dynamic vessel-like structures were observed and they might correspond to high metabolic activity in the periportal/perivenous zones. In the 1-week NAFLD model, a macroscopic, ring-shaped dynamic structure was observed which may indicate highly-motile lipid droplets. In the 2-week NAFLD model, highly-dynamic fragmented vessel-like structures were observed, which are believed to correspond to inflammatory cells around large vessels. In the normal mouse kidney, superficial pipe-like metabolic structures were observed that may correspond to the kidney renal tubules. The pipe-like structures did not appear in the obstructed kidney model.
In the biological and pharmaceutical research, there is a certain demand for label-free three-dimensional (3D) imaging of tissue function. Dynamic optical coherence tomography (OCT) has been demonstrated to fill this demand, but a very high-speed system is required for a volume acquisition of dynamic OCT. Here we demonstrate two new frame acquisition protocols which potentially enable the volumetric dynamic OCT by standard-speed OCT device with a reasonably short measurement time. One of the protocols takes multiple frames at a single location with a few hundred milliseconds interval. The other sequentially takes four frames at a single location, and then repeats this burst four-frame acquisition eight times with a few hundred milliseconds interval. These protocols were validated with in vitro and ex vivo samples. Both protocols gave reasonable dynamic OCT images.
We present a 3-D non-invasive OCT-based tissue dynamics imaging method to evaluate the tumor spheroid drug response. Our method depends on newly developed 3-D scanning protocol, which acquires the volumetric tomography in 52.4 s. The scanning protocol repeats raster scanning 32 times at each location in the tissue in 6.55 s. The tissue sub-cellular motion/viability is quantified by analyzing the OCT time sequence using our developed algorithms including “logarithmic intensity variance algorithm (LIV)” and “late OCT correlation decay speed (OCDSl)”. The capability of our method has been investigated by evaluating the response of the human originated breast cancer (MCF-7) and colon cancer (HT-29) spheroids to anti-cancer drugs. The tissue viability alterations induced by the drug applications have been successfully visualized and quantified.
We present 3D intracellular motility imaging in MCD-diet induced non-alcoholic fatty liver disease (NAFLD) model by OCT-based dynamics imaging method, logarithmic intensity variance (LIV). LIV imaging visualizes the label-free intracellular activity. A 1-week and 2-week NAFLD model were investigated. In 1-week NAFLD, formation of large number of highly dynamic small particles at the beneath of the tissue surface were observed in LIV volume rendering image. In 2-week NAFLD model, a thin high LIV layer signal appeared in cross-sectional LIV image just beneath the tissue surface. The LIV projection and volume rendering images also reveal several discontinuous vessel-like structures.
We demonstrate label-free imaging of renal function with a unilateral ureteral obstruction (UUO) kidney mouse model. The imaging was performed by optical coherence microscopy which is capable of measuring tissue dynamics. Two different studies comprising of 1-week and 2-week UUO models were performed. A circular ring-shape high dynamics appearance at the periphery of the tissue surface was found in the 1-week UUO model for both obstructed and contralateral non-obstructed kidneys. In the 2-week UUO model, several vertical high dynamics regions were observed in cross-sectional dynamics images for both obstructed and non-obstructed kidneys. The results were validated by histological analysis.
We present a non-invasive (label-free) and 3-D method for anti-cancer drug response evaluation of human originated tumor spheroids. Our method is a combination of an OCT microscope and a statistical analysis framework of the OCT signal temporal fluctuations induced by the intra-cellular motions. This method allows the visualization of the 3-D metabolic activity of tumor spheroids and its alteration induced by the drug applications. Two human originated tumor spheroid’s drug responses were evaluated including breast adenocarcinoma (MCF7 cell-line) and colorectal carcinoma (HT-29 cell-line). The drug response was not only visualized as an images, but also quantitative analysis has been performed.
A multi-functional optical coherence microscopy capable of computational refocusing, tissue dynamics and birefringence imaging, and scatterer density estimation is demonstrated. It is applied to cell spheroid, ex vivo animal tissues.
A three-dimensional multi-contrast tissue dynamics imaging method based on polarization-sensitive optical coherence tomography is presented to visualize microvascular tissue activity of mouse livers. Temporal variance of birefringence, temporal polarization uniformity and logarithmic OCT intensity variance are used to access the tissue dynamics. These methods are applied to time-course microvasculature activity visualization of dissected normal and inflammatory mouse liver. Multi-contrast projection images are generated to visualize vascular network of the liver. Cross-sectional and en face dynamics images show high activity around the periportal region of mouse liver at initial time point. Degradation of tissue activity is demonstrated by time-lapse imaging.
We present a new no-invasive label-free OCT-based tissue dynamics/subcellular motion imaging method to visualize and quantify the tissue activity of tumor spheroid. Our method is based on the statistical analysis of the OCT intensity fluctuations of the rapidly acquired OCT signals. The analysis includes log intensity variance (LIV) and OCT correlation decay speed (OCDS). The presented methods have been utilized to visualize and quantify the necrotic activity of the human originated tumor spheroids along 20 hours as cross-sectional and 3-D tomography. This necrotic activity of the spheroid has been not only visualized as an image, but also quantification of the necrotic cell ratio in the spheroid region has been presented.
Optical coherence tomography (OCT) has been widely used for imaging biological sample due to its capability of three-dimensional (3-D) reconstruction. Recently, polarization-sensitive optical coherence tomography (PSOCT) has been used to investigate polarization properties of samples such as retina and muscle [1,2]. Such extensions provide additional contrasts other than traditional reflected intensity and offer a 3-D multi-functional reconstruction of materials and biological tissue. PS-OCT uses A-scan wise computation to obtain the polarization properties from a set of OCT images. In this computation, the lateral resolution is implicitly assumed as infinitely high. However, the birefringence measurement itself will be affected by the lateral optical resolution, defocus, and aberrations. One simple way to obtain high-resolution birefringent data is to use an objective with higher NA. However, OCT also suffers from the trade-off between the lateral resolution and the depth-of-focus (DOF), which might limit its application in thick samples. There have been various methods to overcome this issue. Hardware solutions such as mechanical depth scanning [3] have been reported, but the additional configurations would increase the complexity of system. On the other hand, computational methods do not require additional hardware set-up and thus can be easily adopted, such as interferometric synthetic aperture microscopy (ISAM) [4] and forward model based computational refocusing [5]. However, to our acknowledgement, there are only few reports of long imaging depth for 3-D birefringence imaging. One presented work combined the ISAM with PS-OCT [6]. But the impact of refocusing on multi-contrast imaging, such as artifact in birefringence measurement, has not been thoroughly investigated. In this paper, we present a DOF extended polarization-sensitive imaging by applying computational refocusing to Jones-matrix based PS-OCT (JM-OCT). Computational refocusing is applied to each of four polarization channels (images) of JM-OCT, and the birefringence and degree-of-polarization-uniformity (DOPU) images are computed from the refocused OCT images. Enhanced lateral resolution away from the focus plane and hence extended DOF are demonstrated through phantom and ex vivo porcine muscle measurements. The ex vivo porcine muscle measurement also suggests that the refocusing may reduce the birefringence artifacts.
We present a new non-invasive label-free imaging method, which visualizes tissue dynamics/sub-cellular motion by analyzing the temporal fluctuation of optical coherence tomography (OCT) signals. Our modality has been utilized for visualization and quantification of the time course necrotic process of human breast adenocarcinoma spheroid (MCF7). The response of MCF7 spheroid against anti-cancer drug has also been investigated. The presented method is quantitative. So, the necrotic process was not only shown by images but the dynamics signal value is also plotted as a function of time. It showed clear degradation of tissue activity by time.
In this paper, we demonstrate polarization-sensitive optical coherence tomography (PS-OCT) based tissue dynamics imaging methods to investigate the activity of mouse livers. The methods include logarithmic intensity variance (LIV), temporal variance of birefringence, temporal degree-of-polarization-uniformity. Portal veins appeared as hypo-scattering structure in static OCT intensity. High dynamics appearance around the portal veins in all the dynamics contrasts was found just after the dissection of mouse liver. Although, high activity around the vessels reduced by time, a bright and high dynamics spot appeared in the vessel in LIV cross-sectional and en face image after 28-hours of the dissection.
Due to asynchronization between the acquisition trigger and K-clock trigger in a swept source optical coherence tomography (SS-OCT) system, trigger jitter causes the spectrum a temporal shift in the spectral domain and thus corrupts the measurement. We study ternary distribution of the jitter signal by measuring TiO2 phantom using a SS-OCT system, and it shows one-pixel spectral shift in the spectral domain.
A new method for quantitative assessment of tissue dynamics and activity is presented. The method is based on polarizationsensitive optical coherence tomography. Temporal variance of birefringence and temporal polarization uniformity are used to assess the tissue dynamics. These methods are applied to hourly time-course evaluation of tissue activity of ex-vivo dissected mouse heart.
We present a new OCT-based tissue dynamics/subcellular motion analysis method to visualize tissue dynamics, where we increase the functionality of OCT to be sensitive for tissue dynamics by utilizing rapid-time-sequence analysis of OCT signals. These analysis includes log intensity variance (LIV) and OCT time-correlation analysis (OCT decorrelation speed; OCDS). In addition to LIV and OCDS methods, attenuation coefficient (AC), birefringence, and degree of polarization uniformity (DOPU) analysis were performed. These methods used to visualize and quantify long-term tissue dynamics degradation of different tissue types such as dissected mouse liver and tumor spheroids. These methods were quantitative, so the time-course tissue dynamics degradation has been not only visualized as an image, but also quantitative analysis of the dynamics degradation were performed.
We examined the relationship between depth-resolved local optical properties of eye-corner skin measured by multifunctional Jones matrix optical coherence tomography (JM-OCT) and corresponding wrinkle morphology of aged women (n=21; age range, 71.7±1.7). Wrinkle morphology parameters were analyzed by measuring surface topography of three-dimensional silicone replicas. The same regions were measured three-dimensionally by JM-OCT and the means of several optical properties were computed at each depth. Optical properties include birefringence (BR), attenuation coefficient (AC), and degree-of-polarization uniformity (DOPU). BR and AC were correlated with mean wrinkle depth (WD), although DOPU was not. Significant correlations were found between WD and BR at 88.2 to 138.6 μm depth region from the skin surface (highest correlation at 113.4 μm), and between WD and AC at 12.6 to 18.9 μm and 189 to 459.9 μm depth regions from the skin surface (highest correlations at 18.9 μm and 415.8 μm). This suggests that the collagen structure of the papillary dermis and the microstructure and/or tissue density of the upper epidermis and reticular dermis may be associated with wrinkle morphology. Multiple regression analysis was used to examine the highest significant correlations of BR (113.4 μm) and AC (18.9 μm, 415.8 μm). A significant regression coefficient (R2=0.547, p = 0.001) was obtained, indicating that only BR and AC could sufficiently explain WD. Beta coefficients of BR (113.4 μm), AC (18.9 μm), and AC (415.8 μm) were −0.384, −0.369, and −0.354, respectively. This suggests that the upper epidermis, papillary dermis, and reticular dermis may contribute similarly to wrinkle formation.
Polarization-sensitive optical coherence elastography was developed. It integrates Jones matrix-based polarization-sensitive optical coherence tomography with compression OCE. The method simultaneously measures OCT, attenuation coefficient, birefringence, and tissue mechanical properties. Ex-vivo porcine esophagus was measured by PS-OCE. Evident alteration caused by heat-induced denaturation was obtained in almost all of the optical and mechanical properties including attenuation coefficient, birefringence, in-plane lateral displacement, and microstructural deformation.
Local statistics are widely utilized for quantification and image processing of OCT. For example, local mean is used to reduce speckle, local variation of polarization state (degree-of-polarization-uniformity (DOPU)) is used to visualize melanin. Conventionally, these statistics are calculated in a rectangle kernel whose size is uniform over the image. However, the fixed size and shape of the kernel result in a tradeoff between image sharpness and statistical accuracy. Superpixel is a cluster of pixels which is generated by grouping image pixels based on the spatial proximity and similarity of signal values. Superpixels have variant size and flexible shapes which preserve the tissue structure. Here we demonstrate a new superpixel method which is tailored for multifunctional Jones matrix OCT (JM-OCT). This new method forms the superpixels by clustering image pixels in a 6-dimensional (6-D) feature space (spatial two dimensions and four dimensions of optical features). All image pixels were clustered based on their spatial proximity and optical feature similarity. The optical features are scattering, OCT-A, birefringence and DOPU. The method is applied to retinal OCT. Generated superpixels preserve the tissue structures such as retinal layers, sclera, vessels, and retinal pigment epithelium. Hence, superpixel can be utilized as a local statistics kernel which would be more suitable than a uniform rectangle kernel. Superpixelized image also can be used for further image processing and analysis. Since it reduces the number of pixels to be analyzed, it reduce the computational cost of such image processing.
A maximum a-posteriori (MAP) estimator for signal amplitude of optical coherence tomography (OCT) is presented. This
estimator provides an accurate and low bias estimation of the correct OCT signal amplitude even at very low signal-tonoise
ratios. As a result, contrast improvement of retinal OCT images is demonstrated. In addition, this estimation method
allows for an estimation reliability to be calculated. By combining the MAP estimator with a previously demonstrated
attenuation imaging algorithm, we present attenuation coefficient images of the retina. From the reliability derived from
the MAP image one can also determine which regions of the attenuation images are unreliable. From Jones matrix OCT
data of the optic nerve head (ONH), we also demonstrate that combining MAP with polarization diversity (PD) OCT
images can generate intensity images with fewer birefringence artifacts, resulting in better attenuation images. Analysis of
the MAP intensity images shows higher image SNR than averaging.
A tissue discrimination algorithm of polarization sensitive optical coherence tomography (PS-OCT) based on optical properties of tissue was developed. We calculated three dimensional (3D) feature vector (intensity, extinction coefficient, birefringence) obtained by PS-OCT. The tissue types of each pixel were discriminated according to positions of the feature vectors in 3D feature space. This algorithm was applied for discrimination of human anterior eye chamber. Conjunctiva, selera, trabecular meshwork (TM), cornea and uvea were well separated in the 3D feature space and discriminated in good contrast.
The dermal degeneration accompanying photoaging is considered to promote skin roughness features such as wrinkles. Our previous study demonstrated that polarization-sensitive spectral domain optical coherence tomography (PS-SD-OCT) enabled noninvasive three-dimensional evaluation of the dermal degeneration of photoaged skin as a change in dermal birefringence, mainly due to collagenous structures. Our purpose is to examine the relationship between dermal birefringence and elasticity and the skin morphology in the eye corner area using PS-SD-OCT. Nineteen healthy male subjects in their seventees were recruited as subjects. A transverse dermal birefringence map, automatically produced by the algorithm, did not show localized changes in the dermal birefringence in the part of the main horizontal wrinkle. The averaged upper dermal birefringence, however, showed depth-dependent correlation with the parameters of skin roughness significantly, suggesting that solar elastosis is a major factor for the progress of wrinkles. Age-dependent parameters of skin elasticity measured with Cutometer did not correlate with the parameters. These results suggest that the analysis of dermal birefringence using PS-SD-OCT enables the evaluation of photoaging-dependent upper dermal degeneration related to the change of skin roughness.
Conjunctiva and sclera are not always discriminated in anterior eye optical coherence tomography (OCT) images, although
they have distinctive tissue properties. In contrast, characteristic pattern can be observed at the sclera in a phase retardation
OCT image measured by polarization sensitive OCT (PS-OCT). This is because sclera consists of collagen and it has
birefringence. We developped a new algorithm which discriminates conjunctiva and sclera based on the local statistics of
intensity and phase retardation OCT images. In 4 of 4 cases, conjunctiva and sclera were discriminated. This new algorithm
is useful to discriminate tissues of PS-OCT images not by anatomical tissue structures but by direct tissue properties.
Noninvasive evaluation of dermal degeneration is important for a diagnosis of photoaging. Polarization sensitive optical coherence tomography (PS-OCT) can measure the distribution of phase retardation caused by birefringence property of collagen. The PS-OCT has been reported as the technology of in vivo investigation of collagen structure in the skin. PS-spectral domain (SD)-OCT provides a three-dimensional phase retardation volume, and it provides an en face distribution map of the slopes of cumulative phase retardation. In this research, we measured the lateral orbital area and forehead of 7 young (age = 24.7±1.1) and 6 elder (age = 72.5±1.4) volunteers by a custom-built 840 nm fiber based PS-SD-OCT. This system can measure the skin birefringence three-dimensionally (5.5 sec for a 3-D volume). There was a significant difference in mean of the distribution of phase retardation slopes of the lateral orbital area between young and old cohorts (young: 0.356±0.0255 degree/μm; elder: 0.256±0.0118 degree/μm). However, there was no significant difference in forehead. On the other hand, the shapes of histogram was not normal distribution. Therefore, we calculated skewness and kurtosis of the distributions of slopes for the analysis. In the orbital area, there were also significant differences between young and old cohorts in skewness (y: 0.813±0.135; e: 1.51+/- 0.177) and kurtosis (y: 2.85±0.483; e: 5.23±0.862). In forehead, there was a significant difference between young and old cohorts in skewness (p=0.0289) in contrast with mean (p= 0.065).
B-scan images of phase retardation provide the change of the phase-retardation slope in the cheek dermis with aging.
Enface-based phase-retardation analysis of elder check suggests that the peripheral domain of infundibula have unique
orientation of collagenous fibers.
A two- and three- dimensional swept source optical coherence tomography (SS-OCT) system is demonstrated. This system uses 1.3 &mgr;m probe band, posesses a depth resolution of 14 &mgr;m, and operated at a sensitivity of 100 dB. This SS-OCT is capable of realtime display of two-dimensional OCT, and can take three-dimensional OCT with the measurement time of 2 s.
28 cases of 25 patients including 6 types of corneal diseases, 4 types of uveal diseases, 1 type of scleral disease and 3
types of glaucoma surgery are examined by this SS-OCT.
Corneal opacity is main pathological condition in many corneal diseases which cause
to loss of Visual acuity. In ocular surface clinics, it is important to assess the relationship
between corneal opacity and visual acuity because of patients Quality of Vision. In this
study, swept-source-OCT (SS-OCT) system was employed for imaging of the corneal
opacity disorder. The purpose of this study is to assess the SS-OCT system for the
corneal opacities as quantitative examination and to evaluate the algorithm of
keratectomy simulation. This algorithm provides the entire corneal opacity map and
demonstrated virtual PTK image with ablation profile.
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