We demonstrate longitudinal drug response imaging of tumor spheroids by integrating a spheroid cultivation chamber and the dynamic optical coherence tomography (DOCT) system. The cultivation chamber supports the spheroids with 5 % of CO2 and a temperature of 37 0C. In contrast to our previous cross-sectional time-course imaging method, this newly integrated system enabled longitudinal time-course imaging of a single sample, and hence enabled measuring large number of time-points of the same spheroid. It successfully revealed the temporal property of human breast cancer (MCF-7 cell-line) spheroid’s response to paclitaxel (PTX) and doxorubicin (DOX) with high-temporal-resolution.
We proposed a neural network to generate volumetric dynamic optical coherence tomography (DOCT) from small-number OCT frames. In this study, we used a DOCT method (i.e., logarithmic OCT intensity variance; LIV) and it is applied to tumor spheroid samples. A U-Net-based NN model was trained to generate a LIV image from only 4 OCT frames. The NN-generated LIV was subjectively and objectively compared with conventional LIV images generated from 32 frames. The comparison showed a high similarity between the NN-generated LIV and the conventional LIV. This NN-based method enabled volumetric DOCT with only 6.55 s acquisition time.
Dynamic optical coherence tomography (DOCT) is a method to visualize intratissue activities by analyzing the time sequence of OCT images. We previously established two DOCT contrasts, logarithmic intensity variance (LIV) and late OCT correlation decay speed (OCDSl), and applied them to several medical and pharmaceutical studies. However, these DOCT contrasts have two problems, which are a measurement time dependency of LIV and a difficulty of interpretation of OCDSl. Here we present a new DOCT algorithm which solves these two problems. The new method first computes several LIV values with multiple time window sizes. This LIV shows a monotonically increasing saturation curve. The saturation level and saturation speed, which are named authentic LIV (ALIV) and swiftness, are obtained by fitting the LIVs with a saturation function. Numerical simulation revealed that ALIV is sensitive to the occupancy of the dynamic scatterers over all dynamic and static scatterers, while swiftness is sensitive to the speed of the dynamic scatterers. According to the principle and experimental results using tumor spheroids, ALIV and swiftness are more quantitative and easier to interpret than our previous DOCT methods.
We demonstrate label-free dynamic optical coherence tomography-based evaluation of tumor spheroids' response to multiple anti-cancer drugs based on different mechanisms. The study involved dynamic imaging of human breast cancer (MCF-7) spheroids treated with tamoxifen citrate (TAM), Paclitaxel (PTX), and doxorubicin (DOX). In addition, fluorescence (FLUO) imaging was performed as a reference. The D-OCT imaging is performed using a custom-built OCT device with a repeated raster scan protocol. Two algorithms, including Logarithmic Intensity Variance (LIV) and late OCT Correlation Decay Speed (OCDSl) were used to visualize the tissue dynamics. The D-OCT visualized the drug type-dependent morphological and tissue-activity response patterns of the tumor spheroid. The different response patterns are clearly explained by the mechanisms of the drugs. The presented results suggest that D-OCT can be a useful tool for anti-cancer drug testing.
Differential interference microscopy (DIC) is a method to obtain the refractive index distribution of a sample as contrast. It is suitable for biological cells, however, DIC can only obtain 2D images from thin samples. Therefore, we introduce a new imaging method, volumetric differential contrast (VDC) imaging using optical coherence tomography (OCT). This method enables getting 3D differential contrast of thick samples. VDC was designed based on the disperse scatterer model (DSM), a new theoretical model of OCT, and obtains differential contrast by complex numerical processing of OCT signal. DSM represents the sample as a spatially distributed refractive index with dispersed random scatterers, and OCT signal was formulated from this model. VDC uses two complex OCT signals, s1 and s2 at two laterally slightly distant positions, and the final image is defined as Im[s1 s2. This signal forms a spatially differential image of the product of the refractive index distribution and the scatterer density. According to the formulation, the size of the differentiation kernel, corresponding to the shear amount of DIC, is proportional to the defocus of the probe beam and the separation between s1 and s2. This method was validated by an in vitro spheroid sample and an in vivo zebrafish sample, measured by spectral domain OCT with a center wavelength of 830 nm. VDC images were obtained from refocused and defocused signals.
Cancers is one of the most fatal diseases threatening the survival rate of humans. The recent advances in the cell culture methods allow cultivation of human-derived tumor cells as a 3-D culture, so-called tumor spheroid, which closely emulates the in vivo solid tumors. Hence, it can be used for anti-cancer drug testing, where the efficacy of anti-cancer drugs is evaluated by the drug-induced morphological and viability alterations of the spheroids. The morphological and tissue viability alterations can be evaluated by using staining histology, bright field microscopy, and fluorescence microscopy. However, these methods have several limitations. First, most of them use chemical labeling and/or tissue slicing, and hence, they are invasive. Second, they are 2-D methods. And hence, volumetric morphology, such as alteration of spheroid volume, cannot be evaluated. Third, their penetration-depth is limited to a few hundred microns. It prevents the imaging of thick tissues, such as spheroid. To overcome the above mentioned limitations, we developed a completely label-free 3-D dynamic optical coherence tomography (D-OCT) method for intracellular motility evaluation of tumor spheroid. To validate the utility of our D-OCT method, we organized several studies, including time-course and drug-response evaluations of human breast cancer (MCF-7 cell-line) spheroids. The 3-D morphological and cell viability alterations of MCF-7 spheroid during time-course and under the application of several anti-cancer drugs have been successfully visualized and quantified using D-OCT. The results suggest that D-OCT might be a useful for tumor spheroid-based drug testing and it might be a useful tool for precision medicine research.
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.
We introduce volumetric differential contrast (VDC) imaging using optical coherence tomography (OCT). This method was designed based on a new theoretical model of OCT, the disperse scatterer model (DSM). VDC gives the differential image of “the product of the refractive index distribution and the scatterer density” through complex numerical processing of OCT signals.
The method was validated by in-vitro and in-vivo samples measured by spectral domain OCT. Differential contrast images with arbitrary shear amount and shear direction were obtained at arbitrary depth positions by a single measurement by numerically applying defocus by holographic signal processing after the signal acquisition.
KEYWORDS: Optical coherence tomography, In vitro testing, 3D modeling, Visualization, Tissues, Stereoscopy, Medical research, Lung, Drug development, Cancer
We demonstrate high-resolution (3.8-µm axial and 4.8-µm lateral) three-dimensional dynamic (D-) OCT imaging by 840-nm spectral domain OCT, and compare it with a low-resolution 1.3-µm D-OCT. The D-OCT images are obtained by logarithmic-intensity-variance method, which is sensitive to the magnitude of signal fluctuation.
Human-induced-pluripotent-stem-cell derived alveolar (lung) organoids and human breast cancer (MCF-7) spheroids were examined. The high-resolution D-OCT revealed the tessellation of high and low dynamics at the matured alveolar epithelium. It is also found that such matured alveolar epithelium exhibits ragged inner surface. For the spheroids, high-scattering spots with low dynamics were observed only in the high-resolution image.
We demonstrate OCT-based intracellular motility imaging method, so-called dynamic-OCT (D-OCT), and its application for tumor spheroid-based drug testing. The volumetric tomography is captured in 52.4 s using our custom-designed scanning protocol, which repeatedly capture 32 frames at each location in the tissue. Two algorithms including logarithmic intensity variance (LIV) and late OCT correlation decay speed (OCDSl) were used for tissue dynamics visualization. The utility of our proposed method is investigated for the comparison of three types of anti-cancer drugs applied to human breast cancer (MCF-7) spheroids. The drug type dependent alterations of cell morphology and viability have been successfully visualized.
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 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 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.
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.
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 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.
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.
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