Skin cancer is the most common form of cancer in North America, and melanoma is the most deadly form of skin cancer. Roughness assessment of epidermis has been shown to be valuable in detecting potential skin neoplasia. However, the existing roughness assessment techniques cannot also provide volumetric information. For greater insight, we propose polarization sensitive optical coherence tomography (PS-OCT) for skin assessment. The intensity channel of OCT visualizes the layered structure and surface roughness profile of skin in 3D. Furthermore, PS-OCT can simultaneously conduct polarization related measurements such as the degree of polarization uniformity (DOPU) in a separate imaging channel. Skin phantoms of different surface roughness ranging from 1 to 68 μm have been studied. It was observed that for rougher surfaces, the roughness can be quantified from the surface profile visible in the intensity channel. In smoother surfaces for which the profile is not sensitive, the DOPU decreases with roughness in a quantifiable correlation. The contrast in the DOPU channel is sensitive to polarization and phase fluctuations. Smoother surfaces tend to maintain the polarization state, whereas the height differences in a rougher surface contribute to larger phase shifts between light waves within the coherence volume, leading to greater depolarization. PS-OCT was also applied to in vivo imaging of human skin. The skin at the palm edge shows lower DOPU compared to the skin on the back of the hand, an indication of greater polarization state modification caused by skin roughness. PS-OCT can provide a comprehensive evaluation of skin, which has great potential for detecting melanoma.
Multimodal multiphoton microscopy (MPM) can provide fast, label-free, non-invasive examination of cells, extracellular matrix, and lipids. Two-photon microscopy (2PM) can detect second harmonic generation (SHG) from fibrillar collagen and striated muscle myosin, whereas two-photon excitation fluorescence (2PEF) can detect intrinsic fluorophores such as NADH from cells. Meanwhile, three-photon microscopy (3PM) can detect third harmonic generation (THG) from lipids and tissue interfaces. We have developed a miniaturized multimodal multiphoton system which can perform label-free two-photon and three-photon imaging. An Er-doped fiber laser delivers fundamental pulses at 1580 nm and 80 fs for exciting THG. SHG and 2PEF are excited at 790 nm via the frequency doubling of 1580 nm pulses. For clinical applications, a compact probe is being developed with single-mode fiber for delivering the femtosecond excitation pulses and multi-mode fiber for collecting the MPM signals. A MEMS mirror performs lateral scanning at up to 4 frames/s. For objective lenses, a miniature aspherical lens (NA=0.64) is compared with a gradient index microobjective (NA=0.8). Shape memory alloy actuator used in smartphone cameras is evaluated for shifting the focal plane to acquire Z-stacks for 3D tissue imaging. High-resolution SHG, 2PEF, and THG images are acquired from biological tissues and show that multimodal MPM endomicroscopy has great potential for clinical applications as an alternative to histology.
A segmentation method based on phase retardation measurements from polarization-sensitive optical coherence tomography (PS-OCT) is developed to differentiate the structural zones of articular cartilage. The organization of collagen matrix in articular cartilage varies over the different structural zones, generating different tissue birefringence. Analyzing the slope of the accumulated phase retardation at different depths can detect the variation in tissue birefringence and be used to segment the structural zones. The method is validated on phantoms composed of layers of different materials. Articular cartilage samples from adult swine are segmented with the method. The characteristics in each segmented zone are also examined by histology and high-resolution second-harmonic generation imaging, showing distinctive properties that match with the anatomical structure of articular cartilage. The segmentation algorithm is also applied on PS-OCT images acquired at multiple illumination angles, where the angular dependence of tissue birefringence in the deep zone is detected. This method offers a noninvasive imaging approach to differentiating the structural zones of articular cartilage, as well as a quantification approach based on the phase retardation measurements of PS-OCT. This method has great potential in studying depth-related progression of cartilage degeneration.
Osteoarthritis (OA) is the most common form of arthritis, where the protective cartilage on the ends of bones wears down over time, causing pain, tenderness, stiffness, loss of flexibility and bone spurs. Degenerative alterations start before cartilage loss happens, which include surface swelling, cartilage fibrillation, and calcification. Detecting the early degenerative alterations can assist the diagnosis of early-stage OA. In this study, two imaging modalities are applied on human hip-joint specimens in ex vivo imaging, including polarization-sensitive optical coherence tomography (PS-OCT) and multiphoton microscopy (MPM). OCT detects the layered tissue structure of cartilage and bone using backscattered light and PS-OCT is a functional extension of OCT. PS-OCT measures tissue birefringence which is sensitive to the orderly organization of collagen in cartilage. MPM can visualize collagen fibers with sub-cellular resolution. Complementary information about cartilage on the cellular and tissue level can be obtained by the multimodal imaging. Using the multimodal system, the variation of the thickness of the cartilage structural zones, abnormal birefringence caused by collagen alterations and fibrillation, and uneven structure resulted from calcification are imaged and quantified. The imaging results show distinctive features of degenerative alterations in the OA specimen, such as uneven tissue surface, fibrillation, and reduced birefringence. It is shown that PS-OCT has great potential in detecting early stage OA.
A multimodal multiphoton microscopy (MPM) is developed to acquire two-photon and three-photon signals simultaneously, including two-photon excitation fluorescence (TPEF), second harmonic generation (SHG), and third harmonic generation (THG). We have developed a miniature multimodal MPM system based on a dual-wavelength Erdoped fiber laser, which includes the fundamental pulse at 1580 nm to excite THG and the frequency-doubled pulse at 790 nm to excite TPEF and SHG. The laser is coupled by a single mode fiber into a miniature MPM imaging probe. Label-free imaging by TPEF, SHG, and THG are demonstrated on biological samples, obtained from intrinsic fluorophores, collagen, lipid and interface.
Frequency-doubled femtosecond Er-doped fiber laser is a low-cost and portable excitation source suitable for multiphoton endoscopy. The frequency-doubled wavelength at 780 nm is used to excite the intrinsic fluorescence signal. The frequency-doubling with a periodically poled MgO : LiNbO3 (PPLN) is integrated in the distal end of the imaging head to achieve fiber connection. The imaging speed is further improved by optimizing the excitation laser source. A 0.3-mm length of PPLN crystal is selected and the Er-doped fiber laser is manipulated to match its bandwidth with the acceptance bandwidth of the PPLN. Through this optimization, a reduced pulsewidth of 80 fs of the frequency-doubled pulse is achieved. All-fiber dispersion compensation and pulse compression by single mode fiber is conducted, which makes the fiber laser directly fiber-coupled to the imaging head. An imaging speed of 4 frames / s is demonstrated on ex vivo imaging of unstained biological tissues, which is 10 times faster than our previous study using a 1-mm-long PPLN. The results show that miniature multiphoton endoscopy using frequency-doubled Er-doped fiber laser has great potential for clinical applications.
Polarization-sensitive optical coherence tomography (PS-OCT) and second harmonic generation (SHG) microscopy are
two imaging modalities with different resolutions, field-of-views (FOV), and contrasts, while they both have the
capability of imaging collagen fibers in biological tissues. PS-OCT can measure the tissue birefringence which is
induced by highly organized fibers while SHG can image the collagen fiber organization with high resolution. Articular
cartilage, with abundant structural collagen fibers, is a suitable sample to study the correlation between PS-OCT and
SHG microscopy. Qualitative conjecture has been made that the phase retardation measured by PS-OCT is affected by
the relationship between the collagen fiber orientation and the illumination direction. Anatomical studies show that the
multilayered architecture of articular cartilage can be divided into four zones from its natural surface to the subchondral
bone: the superficial zone, the middle zone, the deep zone, and the calcified zone. The different zones have different
collagen fiber orientations, which can be studied by the different slopes in the cumulative phase retardation in PS-OCT.
An algorithm is developed based on the quantitative analysis of PS-OCT phase retardation images to analyze the
microstructural features in swine articular cartilage tissues. This algorithm utilizes the depth-dependent slope changing
of phase retardation A-lines to segment structural layers. The results show good consistency with the knowledge of
cartilage morphology and correlation with the SHG images measured at selected depth locations. The correlation
between PS-OCT and SHG microscopy shows that PS-OCT has the potential to analyze both the macro and micro
characteristics of biological tissues with abundant collagen fibers and other materials that may cause birefringence.
We report on the development of a compact multiphoton microscopy (MPM) system based on a frequency-doubled, femtosecond erbium-doped fiber laser source at 1.58 μm. By use of periodically poled MgO:LiNbO3, frequency-doubled pulses at 790 nm with average power of 75 mW and pulse width of 130 fs are applied as the excitation source. The fiber laser is optimized for its parameters along with the dispersive properties of the delivery fiber such that the MPM signal is maximized at the sample location. Micro-electro-mechanical system (MEMS) scanner, miniature objective, and multimode fiber are further used to make the MPM system compact. MPM images are obtained from unstained biological samples. The MPM system with a compact, portable, low-cost fiber laser has a great potential to transform the bench-top MPM system to a portable system for in vivo MPM imaging.
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