Technology Development

Space-division multiplexing optical coherence tomography (SDM-OCT)

Ref: Zhou, Chao, et al. “Space-division multiplexing optical coherence tomography.” Optics express 21.16 (2013): 19219-19227.

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. 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. Our 1st generation SDM-OCT system was developed based on fiber optics. However, it required extensive efforts to assemble fiber components (optical splitter, optical delay, fiber array, in the red rectangle of figure A) and control optical delays between different channels by hand, which made it challenging for mass-reproduction. As a solution, we seek to integrate all these fiber components into a photonic chip and incorporate it into our 2nd generation SDM-OCT system. Optical delays and spacing between each output beam of the chip can be customized and fabricated precisely with sub-micron tolerances. The SDM technology provides a new orthogonal dimension for further speed improvement for OCT with favorable cost scaling. With integrated photonic chips, cost scaling will be further reduced, which facilitates broad dissemination of the SDM-OCT technology. SDM-OCT also preserves image resolution and allows synchronized cross-sectional and three-dimensional (3D) imaging of biological samples, enabling new biomedical applications.

Figure: Concept for SDM-OCT. a). Schematic diagram of the prototype SDM-OCT system. The key to the technology is to create multiple illumination beams on the sample simultaneously while having different optical delays for each beam (see the red rectangular region). A single detection channel was used to collect signals from all beams simultaneously. b). A 1×8 fiber array with 300 μm spacing between individual fibers was used in the prototype system. c). Each beam was optically delayed. Signals from different beams (different sample locations) were presented at different frequency range (i.e. imaging depth). For simplicity, only 4 beams are shown. d). Fiber optical components (in the red rectangle of figure A) are integrated into a photonic chip. Optical delay and spacing between each output beam of the chip can be precisely defined.

Ref: Huang, Yongyang, et al. “Wide-field high-speed space-division multiplexing optical coherence tomography using an integrated photonic device.” Biomedical optics express 8.8 (2017): 3856-3867.

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 that can be reliably manufactured with high precisions and low per-unit cost. A three-layer cascade of 1 × 2 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.

Figure: (A) Schematic arrangement of SDM-OCT setup with an integrated photonic chip. (B) Layout of the photonic chip. Input and output of the chip was labeled. A three-layer cascade of 1 × 2 splitters were shown. Red dashed lines indicate 3 layers of 1×2 splitters, respectively. After 3 layers of splitters, the incident light was split from 1 to 8 beams. Optical delay (ΔL) was ~2.5 mm on the chip. (C) Zoom-in view of output port of the chip showing eight waveguide channels with a d = 0.25 mm spacing between them. Output channels were 8° angle polished to reduce back reflections. (D) Photograph of the chip. The chip measures 2.5 × 2.0 cm2, close to the size of a US quarter coin. (E) Roll-off measurement of the central beam of the chip-based SDM-OCT in logarithmic scale. Imaging depth range of the system was measured to be ~31.6 mm in air. A roll-off of ~2dB was observed over ~27 mm depth range in air. (F) Transverse resolution was measured to be ~20 μm with a USAF target (Group 4, element 5 is clearly visible). C: Circulator; C1, C2: collimator; DBD1, DBD2: dual balanced detectors; L1, L2, L3: lenses; M1: mirror; PC: polarization controller.

Molecular-targeted OCT and OCM for cancer detection

Ref: Zhou, Chao, et al. “Photothermal optical coherence tomography in ex vivo human breast tissues using gold nanoshells.” Optics letters 35.5 (2010): 700-702.

OCT is a powerful tool for assessing tissue architectural morphology. It enables 3D imaging with resolutions approaching standard histopathology (a few microns), and it can be performed in vivo and in real-time without the need to remove and process specimens. OCM combines coherence-gated detection with confocal microscopy in order to achieve high transverse resolutions, thus enabling 3D visualization of cellular features. However, current OCT and OCM imaging technologies have not been able to leverage the recent advances in molecular-targeted contrast agents that are revolutionizing biomedicine. In this project, we will develop and validate techniques that enable molecular contrast for 3D-OCT and OCM. The successful completion of this project will allow both the structure and pathological states of tissue to be imaged in 3D, in vivo, and in real time with micron-level spatial resolutions at multiple scales. This work will lay the foundation for a wide range of fundamental research, small animal imaging, and future clinical applications in humans. This work will also serve as a starting point for the OCT and OCM studies of other pathologies associated with abnormal protein expression levels, such as neurodegenerative and cardiovascular diseases. This work is supported by NIH/NIBIB through the Pathway to Independence Award (K99/R00).

Figure: Schematic of photothermal OCT setup. A collinear photothermal excitation beam is coupled into a swept source OCT imaging system.


Optogenetic pacing in Drosophila melanogaster using integrated OCM imaging and red light stimulation system

Ref: Alex, Aneesh, et al. “Optogenetic pacing in Drosophila melanogaster.” Science advances 1.9 (2015): e1500639.

Electrical stimulation is currently the gold standard for cardiac pacing. However, it is invasive and nonspecific for cardiac tissues. We recently developed a noninvasive cardiac pacing technique using optogenetic tools, which are widely used in neuroscience. Optogenetic pacing of the heart provides high spatial and temporal precisions, is specific for cardiac tissues, avoids artifacts associated with electrical stimulation, and therefore promises to be a powerful tool in basic cardiac research. We demonstrated optogenetic control of heart rhythm in a well-established model organism, Drosophila melanogaster. We developed transgenic flies expressing a light-gated cation channel, channelrhodopsin-2 (ChR2), specifically in their hearts and demonstrated successful optogenetic pacing of ChR2-expressing Drosophila at different developmental stages, including the larva, pupa, and adult stages. A high-speed and ultrahigh-resolution optical coherence microscopy imaging system that is capable of providing images at a rate of 130 frames/s with axial and transverse resolutions of 1.5 and 3.9 µm, respectively, was used to noninvasively monitor Drosophila cardiac function and its response to pacing stimulation. The development of a noninvasive integrated optical pacing and imaging system provides a novel platform for performing research studies in developmental cardiology.

Figure: Optogenetic pacing of the Drosophila heart. (A) Schematic of the integrated OCM imaging and pacing system. The optogenetic excitation beam was coupled with the sample arm of the spectral domain OCM system using a dichroic beam splitter. (B) Comparison of cardiac-specific mCherry fluorescence expression between ChR2-expressing transgenic flies (24B-GAL4;UAS-H134R-ChR2) and control flies (24B-GAL4/+). (C) M-mode images showing optogenetic pacing in ChR2 and control adult flies. The ChR2 fly heart with an RHR of 6 Hz was successfully paced at three different frequencies: 8, 9, and 10 Hz. In comparison, the control fly heart was not responsive to optical pacing stimulations.

Developmental Biology

Ref: Alex, Aneesh, et al. “A circadian clock gene, Cry, affects heart morphogenesis and function in Drosophila as revealed by optical coherence microscopy.” PloS one 10.9 (2015): e0137236.

Circadian rhythms are endogenous, entrainable oscillations of physical, mental and behavioral processes in response to local environmental cues such as daylight, which are present in the living beings, including humans. Circadian rhythms have been related to cardiovascular function and pathology. However, the role that circadian clock genes play in heart development and function in a whole animal in vivo are poorly understood. The Drosophila cryptochrome (dCry) is a circadian clock gene that encodes a major component of the circadian clock negative feedback loop. In this study, we utilized ultrahigh-resolution optical coherence microscopy (OCM) system to perform non-invasive and longitudinal analysis of functional and morphological changes in the Drosophila heart throughout its post-embryonic lifecycle for the first time. The Drosophila heart exhibited major morphological and functional alterations during its development. In order to study the functional role of dCry on Drosophila heart development, we silenced dCry by RNAi in the Drosophila heart and mesoderm, and quantitatively measured heart morphology and function in those flies throughout its development. Silencing of dCry resulted in slower HR, reduced CAP, smaller heart chamber size, pupal lethality and disrupted posterior segmentation that was related to increased expression of a posterior compartment protein, wingless. Collectively, our studies provided novel evidence that the circadian clock gene, dCry, plays an essential role in heart morphogenesis and function.

Figure: 3D and M-mode OCM imaging of post-embryonic Drosophila lifecycle. (a) 3D OCM renderings of a 24B-GAL4/+ Drosophila flies at larva, pupa and adult stages. (b) Axial OCM sections showing heart remodelling during Drosophila lifecycle. * denotes the air bubble location during early hours of pupa development. (c) M-mode images at different developmental stages showing HR changes across lifecycle. (d) Examples demonstrating cardiac activity period (CAP) calculation. (e, f) Quantitative analysis of heart rate (HR) and cardiac activity period (CAP) in 24B-GAL4/+ and UAS-dCry-RNAi; 24B-GAL4 flies at different developmental stages.Scale bars in (b) represent 500 μm.

OCT(OCM) combined with deep learning

Ref: Duan, Lian, et al. “Segmentation of Drosophila heart in optical coherence microscopy images using convolutional neural networks.” Journal of biophotonics11.12 (2018): e201800146.

Deep learning is a powerful tool for image processing, such as segmentation and classification. Recently, we have researched and developed deep learning methods for OCT image processing, such as using CNN network to identify and segment the heart region of Drosophila at different developmental stages in the cross-sectional images acquired by a custom 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%. 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. And also, to get a more robust segmentation model, we are now trying to train a LSTM model adding time information in it. What’s more, we are devoted to OCT images denoising using GAN network.

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.

Optical coherence tomography detects necrotic regions and volumetrically quantifies multicellular tumor spheroids

Ref: Huang, Yongyang, et al. “Optical coherence tomography detects necrotic regions and volumetrically quantifies multicellular tumor spheroids.” Cancer research 77.21 (2017): 6011-6020.

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.

3D OCT imaging of Brain Function

Introduction: We will develop novel OCT imaging techniques to image 3D brain functions in animal models. Not only can OCT provide structural information of the animal cortex at micron-scale resolution, but can also be used to extract 3D cerebral hemodynamic information by using Doppler (for blood flow) and spectroscopic (for blood oxygenation) OCT techniques. The 1-2 mm penetration depth of OCT allows imaging through thinned skull rather than opened skull, which makes longitudinal studies possible. The combination of 3D mapping of blood flow and oxygenation will enable for the first time imaging of cerebral oxygen metabolism in 3D at micron-scale resolution. The successful completion of the development of this technique will enable us to investigate 3D brain functions in physiological (e.g. during forepaw, hind paw and whisker stimulations), and pathophysiological (eg. cortical spreading depression, ischemic and traumatic brain injuries) conditions in animal models.

Ref: Li, Fengqiang, et al. “Nondestructive evaluation of progressive neuronal changes in organotypic rat hippocampal slice cultures using ultrahigh-resolution optical coherence microscopy.” Neurophotonics 1.2 (2014): 025002.

Three-dimensional tissue cultures have been used as effective models for studying different diseases, including epilepsy. High-throughput, nondestructive techniques are essential for rapid assessment of disease-related processes, such as progressive cell death. An ultrahigh-resolution optical coherence microscopy (UHR-OCM) system with ∼1.5  μm axial resolution and ∼2.3  μm transverse resolution was developed to evaluate seizure-induced neuronal injury in organotypic rat hippocampal cultures. The capability of UHR-OCM to visualize cells in neural tissue was confirmed by comparison of UHR-OCM images with confocal immunostained images of the same cultures. In order to evaluate the progression of neuronal injury, UHR-OCM images were obtained from cultures on 7, 14, 21, and 28 days in vitro (DIVs). In comparison to DIV 7, statistically significant reductions in three-dimensional cell count and culture thickness from UHR-OCM images were observed on subsequent time points. In cultures treated with kynurenic acid, significantly less reduction in cell count and culture thickness was observed compared to the control specimens. These results demonstrate the capability of UHR-OCM to perform rapid, label-free, and nondestructive evaluation of neuronal death in organotypic hippocampal cultures. UHR-OCM, in combination with three-dimensional tissue cultures, can potentially prove to be a promising tool for high-throughput screening of drugs targeting various disorders.

Figure: Representative OCM images obtained from different cultures on (a) DIV 7, (b) 14, (e) 21, and (f) 28, respectively. The representative OCM images were obtained from a depth of ∼50  μm from the slice surface. (c), (d), (g), and (h) are the magnified images of the brown rectangular regions in (a), (b), (e), and (f), respectively. Viable neurons are marked as red dots. (i) Total viable neuron counts from the first 150  μm of the organotypic hippocampal cultures from different DIVs. ***p<0.001. Scale bars: 400  μm in (a), (b), (e), and (f), 200  μm in (c), (d), (g), and (h).