RESEARCH

Ref: C. Zhou, et al., Space-division multiplexing optical coherence tomography, Opt. Express, 21(16), 19219-19227, 2013.

High speed, high resolution and high sensitivity are desirable for optical coherence tomography (OCT). We demonstrate space-division multiplexing (SDM) technology that translates long coherence length of a commercially available wavelength tunable laser into high OCT imaging speed (parallel OCT). In our 1st generation SDM-OCT system, we achieved an effective 800,000 A-scans/s imaging speed using a 100,000 Hz tunable vertical cavity surface-emitting laser (VCSEL). A sensitivity of 94.6 dB and a roll-off of < 2 dB over ~30 mm imaging depth were measured from a single channel. An axial resolution of ~11 μm in the air (or ~8.3 μm in tissue) was achieved throughout the entire depth range.

Ref: J. Jerwick, et al., Wide-field Ophthalmic Space-Division Multiplexing Optical Coherence Tomography, Photonics Research, 8(4), 539-547, 2020.

With the high-speed SDM-OCT imaging technology, we demonstrate wide-field retinal OCT imaging working around 1060 nm, acquired at an effective A-scan rate of 800,000 A-scans/s with volumetric images covering up to 12.5 mm × 7.4 mm on the retina and captured in less than 1 s. A clinical feasibility study was conducted to compare the ophthalmic SDM-OCT with commercial OCT systems, illustrating the high-speed capability of SDM-OCT in a clinical setting.

Ref: Y. Huang, et al., Full-Range Space-Division Multiplexing Optical Coherence Tomography Angiography, 11(8), 4817-4834, 2020.

In standard FD-OCT, A-scan signals are detected as spectral fringes based on wavelength. Post-processing involves reconstructing sample intensity profiles via inverse Fourier transform (IFT) along the depth. Since detected spectral fringes contain real values only, their IFT results are Hermitian. When sample signals range across the zero delay, they will overlap with their complex conjugates, causing the unwanted blurring of the sample signals, denoted as complex conjugate ambiguity. Full-range techniques reconstruct complex fringes, eliminating conjugate terms, and then double the whole imaging range. We demonstrated a full-range space-division multiplexing optical coherence tomography (FR-SDM-OCT) system, utilizing the galvanometer-based phase modulation full-range technique. We demonstrated the feasibility of FR-SDM-OCT angiography to perform simultaneous acquisition of human fingernail angiograms from four positions. FR-SDM-OCT creates new opportunities to apply SDM-OCT to obtain wide-field angiography of in vivo tissue samples free of labeling.

Ref: Y. Huang, et al., Wide-field high-speed space-division multiplexing optical coherence tomography using an integrated photonic device, Biomed. Opt. Express, 8(8), 3856-3867, 2017.

Space-division multiplexing optical coherence tomography (SDM-OCT) is a recently developed parallel OCT imaging method in order to achieve multi-fold speed improvement. However, the assembly of fiber optics components used in the first prototype system was labor-intensive and susceptible to errors. Here, we demonstrate a high-speed SDM-OCT system using an integrated photonic chip working at around 1310 nm that can be reliably manufactured with high precisions and low per-unit cost. A three-layer cascade of 1 × 2 beam splitters was integrated in the photonic chip to split the incident light into 8 parallel imaging channels with ~3.7 mm optical delay in air between each channel. High-speed imaging (~1s/volume) of porcine eyes ex vivo and wide-field imaging (~18.0 × 14.3 mm2) of human fingers in vivo were demonstrated with the chip-based SDM-OCT system.

Ref: S. Hao, et al., Dual-modality imaging system for monitoring human heart organoids beating in vitro, Opt. Lett., 48(15), 3929–3932, 2023.

To reveal the three-dimensional microstructure and calcium dynamics of human heart organoids (hHOs), we developed a dual-modality imaging system combining the advantages of optical coherence tomography (OCT) and fluorescence microscopy. OCT provides high-resolution volumetric structural information, while fluorescence imaging indicates the electrophysiology of the hHOs’ beating behavior. We verified that concurrent OCT motion mode (M-mode) and calcium imaging retrieved the same beating pattern from the heart organoids. We further applied dynamic contrast OCT (DyC-OCT) analysis to strengthen the verification and localize the beating clusters inside the hHOs. This imaging platform provides a powerful tool for studying and assessing hHOs in vitro, with potential applications in disease modeling and drug screening.

Ref: S. Hao, et al., High dynamic range 3D motion tracking using circular scans with optical coherence tomography, Biomed. Opt. Express, 14, 3881–3898, 2023.

Motion artifacts, from such sources as heartbeats, respiration, or peristalsis, often degrade microscopic images or videos of live subjects. We have developed a method using circular optical coherence tomography (OCT) scans to track the transverse and axial motion of biological samples at speeds ranging from several micrometers per second to several centimeters per second. We achieve fast and high-precision measurements of the magnitude and direction of the sample’s motion by adaptively controlling the circular scan pattern settings and applying interframe and intraframe analyses. These measurements are the basis of active motion compensation via feedback control for future in vivo microscopic and macroscopic imaging applications.

Human heart organoids (hHOs) have emerged as a groundbreaking tool in developmental biology and regenerative medicine. These miniature, self-assembled heart-like structures, derived from pluripotent stem cells, enable researchers to mimic human heart development and study congenital heart defects, which are the most common birth defects in humans. The ability to model cardiac development and dysfunction in vitro is invaluable, offering insights into the intricate processes of heart formation, disease mechanisms, and potential drug testing. Heart organoids bridge the gap between simplistic cell cultures and complex animal models, providing a more accurate representation of the human heart’s structural and functional intricacies. Consequently, they hold immense promise for advancing our understanding of cardiac biology and embryology, ultimately contributing to innovative approaches for tackling heart-related developmental disorders and diseases.

Optical Coherence Tomography (OCT) plays a pivotal role in monitoring the growth and development of heart organoids. Its non-invasive, high-resolution imaging capabilities allow researchers to track the structural and functional changes within these miniaturized hearts in real-time. OCT offers a unique advantage by providing label-free, 3D imaging, enabling the observation of organoid growth, chamber formation, and beating patterns. This technology contributes to the precision and efficiency of organoid analysis, enhancing our ability to study heart development and disease mechanisms in an in vitro setting.

Our lab has been progressively working on connecting OCT imaging with heart organoid development through our collaborative efforts.

Ref: Y. R. Lewis-Israeli, et al, C. Zhou and A. Aguirre, Self-assembling Human Heart Organoids for the Modeling of Cardiac Development and Congenital Heart Disease, Nature Communications, 12, 5142, 2021.

In the paper published in 2021, we generated self-assembled human heart organoids from pluripotent stem cells. These organoids closely resemble human fetal cardiac tissues in terms of transcriptomic, structural, and cellular characteristics. They develop complex cardiac features, including internal chambers, multi-lineage cardiac cell types, and vasculature. Figure X-1 demonstrates various aspects of heart organoid development and function, including structural features, electrophysiology recordings, and calcium transients. Moreover, these heart organoids can mimic metabolic disorders associated with congenital heart defects. This research provides an essential tool for studying cardiac development and congenital heart diseases in vitro. It offers valuable insights into heart morphogenesis and paves the way for potential drug testing and disease modeling in a highly accurate and controlled system.

Ref: Y. Ming, et al, Longitudinal Morphological and Functional Characterization of Human Heart Organoids Using Optical Coherence Tomography, Biosensors and Bioelectronics, 114136, 2022.

Subsequent to the initial publication, our laboratory embarked on the construction of human heart organoids (hHOs) and placed a specific focus on the longitudinal examination of their morphological and functional attributes. We harnessed the capabilities of a customized spectral-domain optical coherence tomography (SD-OCT) system to closely monitor the growth of these hHOs. Our observations through OCT, coupled with calcium imaging, enabled us to visualize the development of chamber structures and beating patterns in these hHOs. The application of OCT provided a rapid, label-free, and non-destructive method to produce comprehensive 3D images, ultimately showcasing the potential of hHOs and OCT as a valuable in vitro platform for exploring heart development and disease mechanisms. Figures X-2 and X-3 utilize OCT imaging to offer detailed 3D renderings and functional characterizations of human heart organoids, shedding light on their structural features, beating patterns, and interbeat intervals.

Ref: B. Volmert, et al., C. Zhou, A. Aguirre, A patterned human primitive heart organoid model generated by pluripotent stem cell self-organization. Nature Communications, 14, 8245 2023.

Recently, another study was under review, aiming to replicate in vivo cardiac development through self-organization and maturation strategies. By mimicking in utero gestation conditions with metabolic and hormonal signals, the research achieves significant transcriptomic, cellular, morphological, and functional changes, resulting in patterned heart-tube-like structures. In Figure X4, the OCT system is utilized for live longitudinal imaging of human heart organoids, revealing the presence of large, interconnected chambers as demonstrated through OCT cross-sectional scans from day 20 to day 30. These organoids encompass diverse cell populations found in the primitive heart tube, and they demonstrate anterior-posterior patterning due to endogenous retinoic acid gradients, mirroring natural cardiac development. This innovative approach provides a potent in vitro model for investigating the roles of different cell types and genes in human heart development, studying congenital heart defects, and advancing the creation of synthetic human hearts.

Ref: R. M. Karvas, et al, C. Zhou, S. Dietmann, and T. W. Theunissen, 3D-cultured blastoids model human embryogenesis from pre-implantation to early gastrulation stages, Cell Stem Cell 30, 1148-1165.e7, 2023.

Our collaborator recently presented a significant advancement in modeling human embryogenesis by utilizing naive human pluripotent stem cells to self-organize into three-dimensional “blastoids.” These blastoids capture key features of post-implantation development, including epiblast lumenogenesis, rapid expansion and diversification of trophoblast lineages, and robust invasion of extravillous trophoblast cells. Extended culture of the blastoids leads to the emergence of embryonic germ layers. Additionally, the study highlights the impact of WNT signaling modulation on the balance between epiblast and trophoblast fates in these post-implantation blastoids. In summary, this research provides a continuous and integrated in vitro model system for studying human embryonic and extraembryonic development, spanning from pre-implantation to early gastrulation stages. Figures X-4 collectively depict the efficient formation of blastoids from naive hPSCs with time course images, immunofluorescence confocal imaging, and OCT imaging. Figure X-4 (E-G) utilized the benefits of OCT imaging, showcasing the top-down and side views of aggregates, 3D reconstruction of OCT images revealing the ICM’s localization within a cavitated blastoid, and quantification of ICM position in individual microwells, offering comprehensive insights into the development of these structures.

Ref: Y. Huang, et al., Optical coherence tomography detects necrotic regions and volumetrically quantifies multicellular tumor spheroids, Cancer Research, 77(21); 601120, 2017.

Three-dimensional (3D) tumor spheroid models have gained increased recognition as important tools in cancer research and anti-cancer drug development. However, currently available imaging approaches employed in high-throughput screening drug discovery platforms e.g. bright field, phase contrast, and fluorescence microscopies, are unable to resolve 3D structures deep inside (>50 μm) tumor spheroids. In this study, we established a label-free, non-invasive optical coherence tomography (OCT) imaging platform to characterize 3D morphological and physiological information of multicellular tumor spheroids (MCTS) growing from ~250 μm up to ~600 μm in height over 21 days. In particular, tumor spheroids of two cell lines glioblastoma (U-87 MG) and colorectal carcinoma (HCT 116) exhibited distinctive evolutions in their geometric shapes at late growth stages. Volumes of MCTS were accurately quantified using a voxel-based approach without presumptions of their geometries. In contrast, conventional diameter-based volume calculations assuming perfect spherical shape resulted in large quantification errors. Furthermore, we successfully detected necrotic regions within these tumor spheroids based on increased intrinsic optical attenuation, suggesting a promising alternative of label-free viability tests in tumor spheroids. Therefore, OCT can serve as a promising imaging modality to characterize morphological and physiological features of MCTS, showing great potential for high-throughput drug screening.

Figure: Determination of necrotic regions of HCT116 tumor spheroids on days 4, 14, and 18 based on intrinsic optical attenuation contrast. The backscattered signals in cross-sectional OCT images (A, E, and I) were used to derive intensity profiles along each axial scan line (B, F, and J). High attenuation regions (indicated by red lines in F and J) could be clearly observed in the intensity profiles of the tumor spheroids on days 14 and 18 (F and J), but not on the tumor spheroid on day 4 (B). Further analyses of optical attenuation coefficient histograms (C, G, and K) were performed to determine the threshold to separate low and high attenuation regions (i.e., 0.48/mm), which is calculated as the median of the two peak values (P1 = 0.36/mm, P2 = 0.60/mm). High-attenuation regions above the threshold highlighted in red (H and L) were detected as the necrotic cores in the tumor spheroids. The region of necrotic tissue clearly increased as the spheroid developed. Scale bars, 100 μm.

Ref: M. Amaral, et al. Three-dimensional imaging and quantification of mouse ovarian follicles via optical coherence tomography. Biomedical Optics Express, 14, 3213-3224, 2023.

Ovarian tissue cryopreservation has been successfully applied worldwide for fertility preservation. Correctly selecting the ovarian tissue with high follicle loading for freezing and reimplantation increases the likelihood of restoring ovarian function, but it is a challenging process. In this work, we explore the use of three-dimensional spectral-domain optical coherence tomography (SD-OCT) to identify different follicular stages, compare the identifications with H&E images, and measure the size and age-related follicular density distribution differences in mice ovaries. We use the thickness of the layers of granulosa cells to differentiate primordial and primary follicles from secondary follicles. The measured dimensions and age-related follicular distribution agree well with histological images and physiological aging. Finally, we apply attenuation coefficient map analyses to significantly improve the image contrast and the contrast-to-noise ratio (p < 0.001), facilitating follicle identification and quantification. We conclude that SD-OCT is a promising method to noninvasively evaluate ovarian follicles for ovarian tissue cryopreservation.

(a) En face projections of an OCT image (left) and stereomicroscope images (center) and OCT renderings (right) of 18-day-old, 4, 12, and 23-week-old mice ovaries. Red arrows point to examples of follicles highlighted by the OCT projection. (b) Three orthogonal views of the 3D OCT dataset: ([AA] top view, [BB] front view, [CC] left view). Scale bar, 500 µm.

Ref: M. Fishman*, A. Matt*, et al., A Drosophila heart optical coherence microscopy dataset for automatic video segmentation, Scientific Data 10, 886, 2023.

Ref: Z. Dong, et al., FlyNet 2.0: Drosophila Heart 3D (2D + time) Segmentation in Optical Coherence Microscopy Images using a Convolutional Long Short-term Memory Neural Network, Biomedical Optics Express, 11(3), 1568-1579, 2020.

Ref: L. Duan, et al., Segmentation on optical coherence microscopy fly heart images using convolutional neural networks, Journal of Biophotonics, e201800146, 2018.

Deep learning is a powerful tool for image processing, such as segmentation and classification. Recently, we have researched and developed deep learning methods for identification and segmentation the heart region of Drosophila at different developmental stages in the cross-sectional images acquired by our customized OCM system. With our well-trained deep learning model, the heart regions through multiple heartbeat cycles can be marked with an intersection over union of ~86%, and further improved to ~92%. Various morphological and dynamical cardiac parameters can be quantified accurately with automatically segmented heart regions. This study demonstrates an efficient heart segmentation method to analyze OCM images of the beating heart in Drosophila.

Figure: Segmentation of Drosophila heart in OCM images using convolutional neural networks.Convolutional neural networks (CNNs) are powerful tools for image segmentation and classification. Here, we use this method to identify and mark the heart region of Drosophila at different developmental stages in the cross-sectional images acquired by a custom optical coherence microscopy (OCM) system. With our well-trained CNN model, the heart regions through multiple heartbeat cycles can be marked with an intersection over union of ~86%. Various morphological and dynamical cardiac parameters can be quantified accurately with automatically seg-mented heart regions. This study demonstrates an efficient heart segmentation method to analyze OCM images of the beating heart in Drosophila.

Ref: Z. Dong, et al., Optical coherence tomography image denoising using a generative adversarial network with speckle modulation, Journal of Biophotonics, e201960135, 2020.

Speckle noise is a key factor affecting OCT image quality. Here, we developed a custom generative adversarial network (GAN) to denoise OCT images. A speckle-modulating OCT (SM-OCT) was built to generate low speckle images to be used as the ground truth. In total, 210 000 SM-OCT images were used for training and validating the neural network model, which we call SM-GAN. The performance of the SM-GAN method was further demonstrated using online benchmark retinal images, 3D OCT images acquired from human fingers and OCT videos of a beating fruit fly heart. The denoise performance of the SM-GAN model was compared to traditional OCT denoising methods and other state-of-the-art deep learning based denoise networks. We conclude that the SM-GAN model presented here can effectively reduce speckle noise in OCT images and videos while maintaining spatial and temporal resolutions.