Shuo Tang

Optical coherence tomography (OCT) is a non-invasive imaging modality that provides high-resolution, three-dimensional images of biological tissue. Since its introduction in the 1990s, it has gained widespread adoption in ophthalmology, and it has also garnered interest in other clinical applications, such as dentistry, endoscopy, and dermatology. More recently, functional extensions of OCT have been developed that allow not only for traditional structural imaging, but also for imaging with functional contrasts in polarization. Skin cancer is a prevalent health burden in the western world, and it is currently diagnosed primarily through an invasive biopsy. As a non-invasive imaging modality, OCT can potentially result in faster skin disease diagnosis and improve quality of care. Studies have shown that OCT can be successful in diagnosing skin cancers such as basal cell carcinoma and squamous cell carcinoma, but traditional OCT has had difficulties in diagnosing melanoma. The further study of OCT’s functional extensions could solve this potential gap.This thesis focuses on Jones matrix polarization-sensitive OCT (PS-OCT), which allows for the simultaneous acquisition of multiple contrasts in intensity, phase retardation, local birefringence, and degree of polarization uniformity (DOPU). Algorithms and methods developed for PS-OCT are explored and show that advanced quantification of skin properties is possible using polarization measurements. We were able to demonstrate that polarization-based measurements are more sensitive to roughness than traditional OCT, and that specific skin layers exhibit unique polarization properties.Finally, A clinical pilot study with 18 healthy volunteers is presented in this thesis. Different skin locations in the body can be differentiated based on their polarization properties, and it is shown that PS-OCT is sensitive to changes related to aging. Additionally, case studies of eczema and vitiligo are examined with PS-OCT, and it is demonstrated that PS-OCT can differentiate features associated with both skin diseases. Our study shows that PS-OCT has high potential for applications in skin disease diagnosis.

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Multiphoton microscopy is a promising imaging technique capable of optical sectioning to acquire images from below the surface of tissue. A miniature objective with depth-scanning capability is needed for clinically adaptation. Imaging performance is dependent upon the ability to focus pulsed laser light. State-of-the-art endoscopes have a depth-scanning capability of ~200 μm and suffer from a significant decrease in resolution at the limit of their depth-scanning range. Zemax software is used to evaluate aspherical lenses for use as objectives, to evaluate the effect of scanning optics, and to design a custom water-dipping objective. Simulations show that the custom objective is capable of achieving diffraction-limited focusing for depths from 0 μm to 1400 μm in water by minimizing spherical aberration. A shape memory alloy actuator is used by the objective for depth-scanning across a 440 μm range, which is experimentally demonstrated by imaging fluorescent beads across the entire range and is more than double the range capability of MPM endoscopes in literature. Resolution measured ~0.7 μm laterally and ~10 μm axially and remained consistent across the entire depth-scanning range. This is a significant improvement to both our lab’s imaging ability and to the depth-scanning abilities of endoscopes presented in literature. Resolution is improved by the addition of corrective optics that reduces axial chromatic aberration. The effect of chromatic aberration on the focusing ability of the custom objective is investigated via simulation using Zemax. Simulations show that the 3 dB wavelengths of the laser’s 80 fs pulses focus at a different depth than the central wavelength and also that marginal rays and on-axis rays arrive at the focal point at different times. By adding CO to reduce chromatic aberration, both of these differences are significantly reduced, which improves the signal to noise ratio. An equation to calculate the difference in arrival time is derived, which shows that the difference is proportional to the square of the objective’s numerical aperture. The deep imaging capabilities of the water-dipping miniature MPM objective is demonstrated by imaging tissue and shows great promise for future clinical use.

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Multiphoton microscopy (MPM) is a promising multimodal imaging technique that can potentially provide fast, label-free examination of cells, extracellular matrices, and lipids similar to histopathology but without the need for biopsy. Each modality provides a complementary view of biological tissue. For example, two-photon microscopy can detect second harmonic generation from collagen and myosin, whereas two-photon excitation fluorescence can detect intrinsic fluorophores such as NADH from cells. Meanwhile, three-photon microscopy can detect third-harmonic generation from lipid-water and other interfaces. On top of deeper penetration and lower background noise when compared to single-photon microscopy, MPM also has inherent optical sectioning capability. However, the adoption of this non-invasive diagnostic technology by hospitals has been slow because most MPM are bulky, expensive, and complex benchtop systems with solid-state lasers that are not suitable for in vivo operations. To translate the technology to clinical applications, a miniature MPM probe or endoscope is required. Our group has previously developed a fiber-based MPM endoscope with a low-cost femtosecond fiber laser. My contribution was to improve and optimize the performance of this system. A key feature missing in past designs was an axial scanning module to enable automatic acquisition of Z-stacks for volumetric imaging. This was addressed by implementing a shape memory alloy autofocus actuator with accurate scanning over 300 microns. Other areas of improvement that were investigated included resolution, excitation power, and collection efficiency for multimodal imaging. Several objective lenses were compared and lateral resolutions below 1 micron and axial resolutions below 10 microns were achieved for field of view between 100 to 200 microns. Different anti-reflection coatings were explored for the relay lenses to increase the transmission power, most notably for third-harmonic generation. Lastly, the position of the multi-mode fiber was simulated and tested to collect a maximum amount of signal for all three contrasts. Ex-vivo Z-stack imaging experiments on leaf, murine, and piscine samples were conducted to demonstrate multimodality and depth scanning. All these additions and enhancements will serve as a stepping-stone for a prototype that will be clinically tested in the future.

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Optical coherence tomography (OCT), the optical analogue of ultrasound with resolution approaching that of histology, has shown great potential in the detection of pre-cancerous and cancerous lesions in the oral cavity. The objective of this thesis is to identify qualitative and quantitative features from OCT that are associated with normal, benign, pre-cancerous and cancerous lesions. In this work, OCT images were acquired from patients in vivo during their visits to the oral pathology clinic. The data were then analyzed both qualitatively and quantitatively. In the qualitative analysis, structural changes in tissue, associated with abnormalities, were compared and matched between their appearance in OCT and the corresponding histology images. In the quantitative analysis, MATLAB was used to automate extraction of numerical values associated with the structural changes, present in the OCT images. As a result of the qualitative analysis, it was found that a set of five features — epithelium thickness, epithelium stratification, rete-ridges visibility, basement membrane presence, and connective tissue appearance relative to the epithelium — can together qualitatively help to identify normal as well as various benign, pre-cancerous and cancerous conditions. To the best of my knowledge, this is the first time that epithelium stratification and high-resolution rete-ridges visibility have been presented as OCT features relevant for cancer detection. Furthermore, quantitative analysis revealed that the numerical values of two of the features — epithelium thickness and stratification — significantly vary going from normal tissue to benign and finally to pre-cancers and cancer. As a result, this work lays the ground work for showing the potential of OCT as a biopsy guidance tool, that can be used in vivo, to find regions of severe abnormalities and thus minimize multiple and unnecessary biopsies.

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Photoacoustic tomography (PAT) is a promising biomedical imaging modality that achieves strong optical contrast and high ultrasound resolution. This technique is based on the photoacoustic (PA) effect which refers to illuminating the tissue by a nanosecond pulsed laser and generating acoustic waves by thermoelastic expansion. By detecting the PA waves, the initial pressure distribution that corresponds to the optical absorption map can be obtained by a reconstruction algorithm. In the linear array transducer based data acquisition system, the PA signals are contaminated with various noises. Also, the reconstruction suffers from artifacts and missing structures due to the limited detection view. We aim to reduce the effect of noise by a denoising preprocessing. The PAT system with a linear array transducer and a parallel data acquisition system (DAQ) has prominent band-shaped noise due to signal interference. The band-shaped noise is treated as a low-rank matrix, and the pure PA signal is treated as a sparse matrix, respectively. Robust principal component analysis (RPCA) algorithm is applied to extract the pure PA signal from the noise contaminated PA measurement. The RPCA approach is conducted on experiment data of different samples. The denoising results are compared with several methods and RPCA is shown to outperform the other methods. It is demonstrated that RPCA is promising in reducing the background noise in PA image reconstruction. We also aim to improve the iterative reconstruction. The variance reduced stochastic gradient descent (VR-SGD) algorithm is implemented in PAT reconstruction. A new forward projection matrix is also developed to more accurately match with the measurement data. Using different evaluation criteria, such as peak signal-to-noise ratio (PSNR), relative root-mean-square of reconstruction error (RRMSE) and line profile comparisons, the reconstructions from various iterative algorithms are compared. The advantages of VR-SGD are demonstrated on both simulation and experimental data. Our results indicate that VR-SGD in combination with the accurate projection matrix can lead to improved reconstruction in a small number of iterations. RPCA denoising and VR-SGD iterative reconstruction have been implemented in PAT. Our results show that RPCA and VR-SGD are promising approaches to improve the image reconstruction quality in PAT.

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Articular cartilage is the connective tissue protecting the joints, which can be divided into four structural zones from shallow to deep: superficial zone, transitional zone, deep zone and calcified zone. Osteoarthritis, a common joint disease, is associated with the progressive degeneration of the cartilage structure. The destruction progressively develops from the superficial zone towards the deep zone as the disease progresses. Thus, visualization of articular cartilage structural zones would significantly facilitate cartilage disease diagnosis, repair, regeneration, and transplantation.Polarization sensitive optical coherence tomography (PS-OCT) is a powerful non-invasive imaging modality capable of evaluating the birefringent properties in biological tissue such as collagen. The second harmonic generation (SHG) imaging in multiphoton microscopy (MPM) can provide complementary high-resolution imaging of the microscopic structure of collagen fibers. In this thesis, we apply both PS-OCT and SHG imaging on articular cartilage. An automated 3-D segmentation method based on PS-OCT phase retardation measurement is developed to differentiate the structural zones of articular cartilage. Since the collagen fiber organization varies from the tissue surface to deep regions, the depth-resolved phase retardation measured by PS-OCT is utilized to distinguish and segment the depth-related structural zones of cartilage tissue. The segmentation results are validated by the high-resolution SHG imaging. This method offers a novel quantification and tissue segmentation approach based on the phase retardation measurement by PS-OCT. A comparison between PS-OCT and SHG imaging on articular cartilage is also carried out. Based on the multilayer architecture of articular cartilage, various features extracted from PS-OCT and SHG are compared along the tissue depth. The segmentation method is implemented to distinguish the tissue layers based on the birefringence property. The segmentation results match well with the different quantification results achieved from the top-view and side-view illumination PS-OCT, and some features extracted from SHG, such as the SHG intensity and the collagen fiber orientation. The results show reasonable association between the tissue birefringence detected by PS-OCT and the fiber organization detected by SHG microscopy. PS-OCT and SHG are capable to analyze both the macro and micro characteristics of collagen fibers in articular cartilage, showing great potential in detecting related disease progression.

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Photoacoustic (PA) imaging is a new biomedical imaging modality that is based on the PA effect. In the PA effect, nanosecond short pulsed laser light illuminates on tissue and is absorbed by optical absorbers, which undergo a temporary temperature rise. The illuminated region experiences thermo-elastic expansion and engenders an abrupt and localized pressure variation. This transient variation causes a PA wave to propagate from the absorber outward through the tissue to the surface for detection by an ultrasound transducer. The detected PA signal can then be used to estimate the initial pressure distribution. PA imaging is an absorption-based modality which is capable of providing the optical property of the illuminated tissue while achieving deep imaging penetration compared with conventional optical imaging. Meanwhile, the PA image can also provide a similar spatial resolution as ultrasound imaging.Photoacoustic tomography (PAT) is the most widely used imaging mode in PA imaging due to its simplicity and versatility. One major drawback in PAT is that the reconstruction algorithm required to estimate the initial pressure demands a large detection view angle for exact reconstruction, which is typically impractical in clinical applications. Our goal of this thesis is to develop a novel methodology to increase the detection view angle by using two linear array transducers at different orientations. The relative position between the two transducers needs to be calibrated in order to combine the received signal from the two transducers. A new calibration approach is developed by using the ultrasound modality. The efficacy of the calibration method is demonstrated by both simulation and experiment. With increased detection view angle, the reconstructed image indicates more complete tissue structures compared with the one acquired by a single transducer.By combining the PAT images from the two transducers, an improvement on the image quality through complementing the structural information is achieved. Our approach does not require a calibration phantom, which can largely simplify the calibration process and shorten the acquisition time. The use of linear array transducers and the flexibility of positioning the transducers to fit tissue geometry make this approach promising for clinical applications.

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Multiphoton microscopy (MPM) and optical coherence tomography (OCT) are two modalities suitable for imaging corneas. MPM is a nonlinear optical imaging technique that has sub-micron spatial resolution, deep penetration, and excellent optical-sectioning capability. It can detect two contrasts simultaneously: two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). TPEF and SHG are used to visualize cells and collagen fibers, respectively. On the other hand, OCT is based on the detection of single-backscattered light using interferometry principles. Generally, it has micrometer-resolution and up to a few millimeters of imaging-depth. Combining MPM and OCT for corneal imaging is intuitive in the current study as the complementary information obtained from cornea helps us to understand the morphological and physiological status of the cornea. Furthermore, the MPM and OCT images can be used to quantitatively characterize the thicknesses and refractive index (RI) of the major corneal layers. The visualization of the corneal morphology is important in ophthalmology. It can be used to: study the effects of contact lens wear and topical medications; diagnose corneal diseases such as infectious keratitis; and observe corneal ulcers, and keratoconous. In addition, the ability to quantitatively characterize the corneal thickness and RI is valuable for many ophthalmologic applications. These two parameters are useful for laser refractive surgeries, and the diagnosis of corneal degeneration, and endothelial dysfunction. They are also linked to other important parameters such as corneal hydration and intraocular pressure. In this thesis, the capabilities of a combined MPM and OCT system for corneal imaging are demonstrated. Firstly, it is used to simultaneously visualize and compare the morphology, and to characterize the thicknesses and the RI of five different species’ corneas. In order to visualize the thicker tissues, the OCT modality is altered in two ways: a prism-based hardware dispersion balancing unit is added to minimize the dispersion mismatch in OCT; and an additional OCT configuration of objective lens and spectrometer setup was introduced. Secondly, the combined system is used to identify an induced parasitic infection in human corneas. The study serves as an important step forward to bring the combined MPM and OCT technology to clinical settings.

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Photoacoustic (PA) imaging is an emerging imaging modality that relies on the PA effect. The PA effect is caused by exposing an optically absorbing sample to near-infrared light which causes the sample to experience a temporary temperature increase through optical absorption. The heated region undergoes thermoelastic expansion and produces an abrupt and localized pressure change. This change results in a transient PA wave that propagates out toward the sample surface for collection by an ultrasound (US) transducer. Through image reconstruction, the optical property of the sample can be obtained.PA imaging is promising in detecting brachytherapy seeds during prostate brachytherapy. The high absorption coefficient of the metallic seeds leads to high PA imaging contrast. One major drawback is the limited imaging depth due to high optical attenuation of the excitation light in tissue. One of the goals of this thesis is to conduct initial feasibility tests of enhancing the PA contrast through brachytherapy seeds modifications. Seed coated with a contrast enhancing material shows an increase of 18 dB in signal-to-noise ratio (SNR) and two time increase in the imaging depth (5 cm). Another method of silver coating leads to a 5 dB improvement in the SNR of the modified seeds. An alternative approach in using dyed ethanol solution as a contrast enhancing agent by filling the spaces between two seeds is also reported. The result showed improvement comparable to the black paint method. Another goal is to propose a novel method of tissue typing in PA imaging. A temperature change in tissue can lead to changes of several tissue parameters which can be used for tissue typing. One of the parameters is the speed of sound in tissue, which increases in water-based non-fatty tissue and decreases in fatty tissue as temperature is raised. We show that on average, 6.9±1.5 %/min increase and 4.2±1.5 %/min decrease in PA intensity are observed in porcine liver and bovine fat samples respectively through one minute of laser heating. These results demonstrate that by analyzing the PA intensity change of the illuminated sample, one can extract characteristic information that can lead to tissue type differentiation.

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Multiphoton Microscopy (MPM) is a widely used imaging technique in the biomedicine field. Fiber-based multiphoton endoscopes are important for in vivo clinical application because they enable minimally invasive imaging. One major challenge for these endoscopes is the efficient delivery of ultrashort pulses in the near infrared region through the optical fibers. MPM requires ultrashort pulses to obtain high peak power for the nonlinear excitation. However, the optical fibers, can introduce dispersion which can severely broaden the pulses and reduce the peak power. The purpose of this study is to find a good candidate of optical fibers that can propagate sub-30 fs pulses while maintaining high peak power for the MPM excitation.In this project, I investigate the feasibility of applying a specially-designed chirped photonic crystal fiber (CPCF) for MPM imaging because CPCF has unique cell-size radial chirp can achieve low dispersion in a broadband transmission window. The key features of the CPCF are characterized, such as spectra, mode profile, and dispersion parameter, etc. A fiber-delivered MPM system is developed by adding the fiber coupling to a multimodal microscope. A prism-based dispersion pre-compensation unit is optimized to compensate the dispersion from all the optical components in the CPCF-delivered system. After pre-compensation, the appealing performance of CPCF applied in MPM is demonstrated by imaging various biological samples. Additionally, traditional hollow core fiber (HCF) is used as a comparison. The HCF consists of several identical reflective layers in the cladding. I pre-compensate the laser pulses after the HCF propagation and MPM images for similar samples are acquired. Large improvement in image contrast is observed in all samples for the system using the CPCF for light delivery compared with the system using the conventional HCF. The enhancement in second harmonic generation (SHG) is more significant than that in two photon excited fluorescence (TPEF).Our study shows that CPCF can successfully deliver sub-30 fs pulses with significantly increased excitation efficiency of MPM for the broad-band laser. These properties are highly sought after in MPM endoscopy. With the fiber delivery of femtosecond pulses, MPM can be developed into a portable system for in vivo imaging.

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Optical coherence tomography (OCT) is a non-invasive optical tomographic technique based on the principle of interferometry. It can capture micrometer-resolution, three-dimensional images of tissues over millimeter field-of-view at a fast speed. Multiphoton microscopy (MPM) is an emerging imaging modality based on the excitation of nonlinear signals from fluorescent molecules and the induction of second harmonic generation (SHG). It is capable of en-face high-resolution imaging with sub-micron resolution. Although OCT and MPM are essential imaging tools for disease diagnosis, each one of them has shortages, such as the low resolution of OCT and the low depth penetration of MPM. The purpose of this study is to design a multimodal imaging system by combining MPM and OCT into a single platform so that the two modalities can complement and overcome the shortages of each other. The design consists of two parts: hardware and software. For hardware, the two modalities are integrated into a single platform, sharing the laser source, the controlling scanners and the sample arm. In addition, the OCT has a reference arm for interference and a custom-built spectrometer for signal detection, whereas the MPM uses two photomultiplier tubes (PMT) for photon detection. For software, two user interfaces are specially designed to control beam scanning and data acquisition of the MPM and OCT respectively.The performance of this mutlimodal system is demonstrated by imaging biological samples. The results indicate that our system is capable of multiscale imaging of multilayered tissues with clear structures. One of the important applications of the multimodal system is measuring the refractive index (RI) and thickness of biological tissues. This capability is demonstrated on fish cornea. The results show our system is capable of imaging as well as quantitative characterization of RI and thickness of multilayered biological tissues. This system can potentially be a powerful tool for disease detection and surgery treatment.

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Spectral domain optical coherence tomography (SD-OCT) is an imaging modality that provides cross-sectional images with micrometer resolution. One major drawback of SD-OCT, however, is the depth dependent sensitivity fall-off by which image quality rapidly degrades in regions corresponding to deeper locations of the sample. This disadvantage is due to the finite spectral resolution of the hardware as well as the software reconstruction method that is used.SD-OCT employs a broadband light source for illumination and a spectrometer for signal detection. This system uses diffraction grating to separate spectral components by wavelengths (λ), which are then detected by a CCD array. The sensitivity fall-off is dependent on the spot size shining on the CCD, with respect to the pixel size of the CCD array. This hardware contribution to the fall-off can be minimized by careful design of the spectrometer. The software reconstruction is based mainly on the discrete Fourier transform (DFT) of the measured spectral data, which can be performed quickly using the widely accepted fast Fourier transform (FFT) algorithm, provided that the input is sampled uniformly in the wavenumber (k) domain. Due to the inverse relationship between k and λ, the data must be resampled to achieve a uniform spacing in k. Accuracy of the resampling method is important for the reconstruction, since the performance of the interpolation algorithm tends to degrade as the signal approaches the Nyquist sampling rate. This also causes a sensitivity fall-off for signals originating at greater depths, which corresponds to a higher modulation fringe frequency in the k domain. The goal of this thesis is to outline the development of a real-time SD-OCT imaging system that can deliver high quality images. The aim is to solve two major problems of current state-of-the-art SD-OCT systems, namely the depth dependent sensitivity fall-off and the image reconstruction time limitation. An SD-OCT system is demonstrated using a new reconstruction approach based on non-uniform fast Fourier transform (NUFFT). Using parallel computing techniques, our system can produce high quality images at over 100 frames per second with less than 12.5dB sensitivity fall-off over the full imaging range of 1.7mm.

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