We propose a B-scan-wise- multi-focus average method (B-MFA) to suppress multiple scattering noise for in-vivo imaging. This method uses only multiple B-scans with modulated focus position for complex averaging, and a one-dimensional computational refocusing is applied to cancel the defocus in each B-scan. To validate the proposed method, ten medaka fish were measured in-vivo without anesthesia. The results show that B-MFA can enhance the OCT image contrast in the deep regions.
We investigate the dependency of dynamic optical coherence tomography (DOCT) signal on wavelength and resolution in two systems. One is 840-nm spectral domain OCT, and the other is 1310-nm swept-source OCT. For wavelength dependency, we made the resolution roughly the same among two systems by computationally reducing the axial resolution of 840-nm system. For resolution dependency, we compared LIV images with and without reduction of the resolution. In addition, we performed numerical simulation by modeling the intracellular scatterer dynamics by a diffusion. Experiment and simulation suggested that LIV is highly affected by the wavelength, where shorter wavelength gives higher LIV.
We investigate wavelength and resolution dependency on dynamic optical coherence tomography (D-OCT) signal using two OCT systems. One is 840-nm spectral domain OCT. The other is 1310-nm swept-source OCT. We kept the resolution roughly the same between the two systems by computationally reducing the axial resolution of 840-nm system. In addition, we performed numerical simulation by modeling the intracellular scatterer dynamics by a diffusion. Experimental results showed that longer wavelengths correspond to lower D-OCT, while lower resolutions lead to slightly lower D-OCT. Simulation results confirm the wavelength dependency of D-OCT, with resolution variations having non-significant impact.
The zebrafish has shown to be an essential preclinical animal model, especially in the field of oncology. A non-invasive, high-resolution, and three-dimensional imaging modality is required to identify disease related changes in this model organism.
The presented polarization-sensitive Jones matrix optical coherence tomography (JM-OCT) prototype was utilized in three different studies, covering in vivo imaging of wildtype zebrafish, an adult postmortem tumor model and a longitudinal xenograft tumor zebrafish investigation.
This work highlights the potential of JM-OCT as a non-invasive, label-free, and three-dimensional imaging tool for preclinical cancer research based on zebrafish models.
Digital refocusing of optical coherence tomography (OCT) restores the spatial resolution degraded by defocusing. To apply this to imaging of biological tissue with point-scan Fourier domain OCT (FD-OCT), fast volume acquisition is needed to prevent motion disturbance. This study demonstrates the Lissajous-cycle-wise (LCW) digital refocusing algorithm applied to in vivo imaging at acquisition rates typical of FD-OCT. Blurring along and perpendicular to the scanning direction are compensated by inverse filtering and compositing A-lines of different scanning directions after motion correction, respectively. Ex vivo and in vivo biological sample experiments are applied for the proof of concept. The blurred images due to defocusing are sharpened by the LCW digital refocus algorithm.
KEYWORDS: Optical coherence tomography, Scattering, Multiple scattering, Glasses, Particles, Tissues, In vivo imaging, Image acquisition, Computation time, 3D acquisition
In this study, we propose a B-scan-wise-multi-focus averaging method (B-MFA) to suppress the multiple scattering noise in optical coherence tomography. This method uses multiple B-scans with different focus positions. These B-scans were one-dimensionally computationally refocused and complex averaged. To verify the multiple scattering noise suppression ability of this method, we measured a phantom consisting of scattering and non-scattering layers. The results show that BMFA suppresses multiple scattering noise.
Optical coherence tomography (OCT) has been utilized to investigate the optical properties of tissue in animal model studies. Polarization sensitive OCT (PS-OCT) is one of the functional extensions of OCT, which provides additional contrasts such as birefringence and degree-of-polarization uniformity (DOPU). It enables multi-contrast imaging for discrimination of tissue properties in biological samples. However, biological samples often exhibit multiple scattering (MS). It severely hampers the penetration depth and degrades the optical property measurement in deep regions for both OCT and PS-OCT. In a recently presented work, it was found that all of the scattering OCT value, birefringence value, and DOPU value were altered if they were measured at the deep region. This alteration is suspected to be artifacts caused by MS. So far, various methods have been proposed to tackle the MS issue. One is averaging several images with decorrelated MS signals. The decorrelation of MS signals was realized by wavefront shaping with complex devices such as spatial light modulators or deformable mirrors. However, these optics increase the cost and system complexity, and the wavefront shaping is still challenging. Simple hardware, such as multimode fiber, was only demonstrated in full-field OCT. In this work, we present a method termed as “multi-focus averaging” (MFA) to suppress the MS effect and improve the image contrast in deep regions of scattering samples. It uses focus modulation by a tunable lens to decorrelate MS signals among multiple acquisitions, and computational refocusing is used to cancel the different defocus during focus modulation. The refocused OCT signals are averaged in complex to reduce MS signals. It is demonstrated in standard OCT scheme, and a scattering phantom and a post-mortem zebrafish are used to validate the intensity contrast improvement. Results show that MFA images can provide better intensity contrast than standard complex averaging in scattering samples. In addition, we further adapt this method to PS-OCT to suppress the MS and to improve the accuracy of polarization sensitive measurement. We found MFA mitigated low-DOPU artifact in polarization sensitive measurement.
The zebrafish is a valuable animal model in pre-clinical cancer research. Optical coherence tomography (OCT) is a non-invasive optical imaging technique, which provides a label-free and three-dimensional method to investigate the tissue structure. Jones-matrix OCT (JM-OCT) is a functional extension of conventional OCT, to gain additional tissue specific contrast by analyzing the polarization states of the back-scattered light. In this work we present the longitudinal investigation of in vivo wildtype and a tumor xenograft zebrafish model using our JM-OCT prototype. The scattering and depth-resolved polarization properties in control versus tumor regions were analyzed and compared to results obtained from histology.
We propose a new multi-focus average method for optical coherence tomography, to reduce the multiple scattering signals and improve the visibility of the sample structure in the deep region. It combines computational refocusing, complex averaging, and multiple acquisitions with focal shifting. A scattering phantom was measured to validate the suppression of multiple scattering signals and the contrast improvement at the deep region.
The zebrafish is an essential animal model in pre-clinical research, especially in the field of cancer investigations. A polarization sensitive Jones matrix OCT (JM-OCT) prototype operating at 1310 nm was utilized to investigate adult control and tumor zebrafish models. Various anatomical features were characterized based on their inherent scattering and polarization properties. A motorized translation stage in combination with the prototype enabled large field-of-view imaging to investigate whole adult zebrafish non-destructively. The reflectivity, the attenuation coefficient and local polarization parameters such as the birefringence and the degree of polarization uniformity were analyzed to quantify differences in tumor versus control regions.
Post-signal-processing techniques of refocusing and digital aberration correction of optical coherence tomography (OCT) restore the spatial resolution deteriorated by optical aberrations. In the case of in vivo biological tissues with Fourierdomain OCT, an ultrafast volumetric acquisition is required to avoid motion distortion. In point-scanning OCT, a fast scan is required, and the bulk phase shifts among surrounding A-lines should be corrected. A low duty cycle might be necessary to obtain the consistent en face image with a high-speed raster scan. Recently, we have demonstrated a Lissajous-patternbased probe beam scanning and motion correction algorithm. In this study, we demonstrate a Lissajous-cycle-wise (LCW) digital refocus algorithm. The algorithm of LCW digital refocus is based on a convolution operation with an inverse filter. The complex OCT signals sampled with a Lissajous cycle are going to be convolved with an inverse filter of defocus. The reconstruction from Lissajous data to the Cartesian coordinate assigns a Cartesian grid to each A-line. By merging A-lines of different cycles, blurring due to defocus perpendicular to the scanning trajectory is mitigated by other cycle data. The phantom experiment is applied for the proof of concept. A prototype 1-μm spectral-domain OCT system is used for experiments. The A-line rate is 92 kHz. The blurred image of a phantom by defocusing is sharpened by the LCW digital refocus process along with all directions in the en face plane. Although the restored resolution does not reach the diffraction limit, an ultrafast volumetric acquisition is not required when one Lissajous cycle is significantly faster than sample motion.
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.
A three-dimensional label-free multi-contrast imaging for ex vivo tissue investigation is presented. Computational refocusing is implemented in a Jones-matrix polarization-sensitive optical coherence tomography (PS-OCT) system to overcome the trade-off between imaging depth and lateral resolution. The application of multiple contrast imaging, including intensity, birefringence, and degree-of-polarization uniformity (DOPU), is demonstrated by phantom, porcine muscle, and zebrafish measurements. Extended imaging depth with enhanced lateral resolution over millimeter is achieved. In tissue imaging we find some altered birefringence and DOPU estimation, whose size and alteration are proportional to defocus amount. This biased estimation can be numerically mitigated after applying computational refocusing.
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.
A polarization sensitive Jones matrix optical coherence tomography (JM-OCT) prototype was utilized to investigate in vivo wildtype zebrafish in different development stages. JM-OCT is a functional extension of conventional intensity-based OCT to visualize polarization properties of samples. We demonstrated simultaneous structural and functional imaging of in vivo wildtype zebrafish at the age of eight-days, one and two-months post-fertilization. The scattering and polarization characteristics of the anatomical features, such as the eye, and the muscles, were investigated and quantified non-invasively and in three-dimensions. Furthermore, the setup was able to visualize dynamic processes like the blood flow in a label-free way.
Significance: The scattering and polarization characteristics of various organs of in vivo wildtype zebrafish in three development stages were investigated using a non-destructive and label-free approach. The presented results showed a promising first step for the usability of Jones-matrix optical coherence tomography (JM-OCT) in zebrafish-based research.
Aim: We aim to visualize and quantify the scatter and polarization signatures of various zebrafish organs for larvae, juvenile, and young adult animals in vivo in a non-invasive and label-free way.
Approach: A custom-built polarization-sensitive JM-OCT setup in combination with a motorized translation stage was utilized to investigate live zebrafish. Depth-resolved scattering (intensity and attenuation coefficient) and polarization (birefringence and degree of polarization uniformity) properties were analyzed. OCT angiography (OCT-A) was utilized to investigate the vasculature label-free and non-destructively.
Results: The scatter and polarization signatures of the zebrafish organs such as the eye, gills, and muscles were investigated. The attenuation coefficient and birefringence changes between 1- and 2-month-old animals were evaluated in selected organs. OCT-A revealed the vasculature of in vivo larvae and juvenile zebrafish in a label-free manner.
Conclusions: JM-OCT offers a rapid, label-free, non-invasive, tissue specific, and three-dimensional imaging tool to investigate in vivo processes in zebrafish in various development stages.
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.
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.
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.
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