Understanding optical reflectance contrast for real-time characterization of epithelial precursor lesions.

Detecting early-stage epithelial cancers and their precursor lesions are challenging as lesions could be subtle and focally or heterogeneously distributed over large mucosal areas. Optical coherence tomography (OCT) that enables wide-field imaging of subsurface microstructures in vivo is a promising screening tool for epithelial diseases. However, its diagnostic capability has not been fully appreciated since the optical reflectance contrast is poorly understood. We investigated the back-scattered intensities from clustered or packed nanometer scale intracellular scatterers using finite-difference time-domain method and 1-µm resolution form of OCT, and uncovered that there existed correlations between the reflectance contrasts and the ultrastructural clustering or packing states of these scatterers, which allows us to interpret the physiological state of the cells. Specifically, both polarized goblet cells and foveolar cells exhibited asymmetric reflectance contrast, but they could be differentiated by the optical intensity of the mucin cup due to the different ultrastructural make-ups of the mucin granules; keratinocytes could demonstrate varied cytoplasmic intensity and their cytoplasmic contrast was closely correlated with the packing state of keratin filaments. Further preliminary study demonstrated that these new understandings of OCT image contrast enables the characterization of precancerous lesions, which could complement the current morphology-based criteria in realizing "virtual histology" and would have a profound impact for the screening and surveillance of epithelial cancers.

Constrained polarization evolution simplifies depth-resolved retardation measurements with polarization-sensitive optical coherence tomography.

We observed that the polarization state of light after round-trip propagation through a birefringent medium frequently aligns with the employed input polarization state 'mirrored' by the horizontal plane of the Poincaré sphere. We explored the predisposition for this mirror state and evidence that it constrains the evolution of polarization states as a function of the round-trip depth into weakly scattering birefringent samples, as measured with polarization-sensitive optical coherence tomography (PS-OCT). Combined with spectral variations in the polarization state transmitted through system components, we demonstrate how this constraint enables measurement of depth-resolved birefringence using only a single input polarization state, which offers a critical simplification compared to conventional PS-OCT employing two input states.

Geometry-Dependent Spectroscopic Contrast in Deep Tissues.

Nano-structures of biological systems can produce diverse spectroscopic effects through interactions with broadband light. Although structured coloration at the surface has been extensively studied, natural spectroscopic contrasts in deep tissues are poorly understood, which may carry valuable information for evaluating the anatomy and function of biological systems. Here we investigated the spectroscopic characteristics of an important geometry in deep tissues at the nanometer scale: packed nano-cylinders, in the near-infrared window, numerically predicted and experimentally proved that transversely oriented and regularly arranged nano-cylinders could selectively backscatter light of the long wavelengths. Notably, we found that the spectroscopic contrast of nanoscale fibrous structures was sensitive to the pressure load, possibly owing to the changes in the orientation, the degree of alignment, and the spacing. To explore the underlying physical basis, we further developed an analytical model based on the radial distribution function in terms of their radius, refractive index, and spatial distribution.

Contrast of nuclei in stratified squamous epithelium in optical coherence tomography images at 800 nm.

Imaging nuclei of keratinocytes in the stratified squamous epithelium has been a subject of intense research since nucleus associated cellular atypia is the key criteria for the screening and diagnosis of epithelial cancers and their precursors. However, keratinocyte nuclei have been reported to be either low scattering or high scattering, so that these inconsistent reports might have led to misinterpretations of optical images, and more importantly, hindered the establishment of optical diagnostic criteria. We disclose that they are generally low scattering in the core using Micro-optical coherence tomography (µOCT) of 1.28-µm axial resolution in vivo; those previously reported "high scattering" or "bright" signals from nuclei are likely from the nucleocytoplasmic boundary, and the low-scattering nuclear cores were missed possibly due to insufficient axial resolutions (~4µm). It is further demonstrated that the high scattering signals may be associated with flattening of nuclei and cytoplasmic glycogen accumulation, which are valuable cytologic hallmarks of cell maturation.

Single input state polarization-sensitive optical coherence tomography with high resolution and polarization distortion correction.

In single input state polarization-sensitive optical coherence tomography (PS-OCT) with high resolution, the imperfections of quarter-wave plate (QWP) and the sensitivity roll-off mismatch between the two detection channels cause unpredictable polarization distortion. We present a correction method based on the Jones matrix modeling of the system. In a single input PS-OCT system working at 840 nm with an axial resolution of ~2.3 µm, the method yielded better estimation of retardation and optic axis orientation with significantly reduced noise level, especially in weakly birefringent samples. Numerical simulations and quantitative imaging of a sample of known birefringence were performed to validate the performance. We further demonstrate the advantages of our approach with birefringence imaging of swine retina, rat aortic wall, and rat esophageal mucosa for potential clinical applications.

Cellular resolution corneal imaging with extended imaging range.

Current optical coherence tomography (OCT) technology, which is used for imaging the eye's anterior segment, has been established as a clinical gold standard for the diagnosis of corneal diseases. However, the cellular resolution level information that is critical for many clinical applications is still not available. The major technical challenges toward cellular resolution OCT imaging are the limited ranging depth and depth of focus (DOF). In this work, we present a novel ultrahigh resolution OCT system that achieves an isotropic spatial resolution of <2 µm in tissue. The proposed system could approximately double the ranging depth and extend the DOF using the dual-spectrometer design and the forward-model based digital refocusing method, respectively. We demonstrate that the novel system is capable of visualizing the full thickness of the pig cornea over the ranging depth of 3.5 mm and the border of the corneal endothelial cells 8 times Rayleigh range away from the focal plane. This technology has the potential to realize cellular resolution corneal imaging in vivo.

Extending the depth of focus of fiber-optic optical coherence tomography using a chromatic dual-focus design.

We report a dual-focus fiber-optic probe designed to extend depth of focus (DOF) in high-resolution endoscopic optical coherence tomography. We exploited the broad spectral bandwidth of a supercontinuum source and, in the fiber probe, the foci of the 750-1000 nm and 1100-1450 nm inputs were axially chromatically shifted. The interference signals from the two spectral bands were measured with a Si camera-based spectrometer and an InGaAs camera-based spectrometer, respectively. We verified the feasibility of the design using a phantom composed of microparticles and swine small intestine tissue ex vivo. The results showed that a transverse resolution below 5 µm over 300 µm could be maintained, and that the extended DOF was 2 times larger than that of the single focus probe via the use of dual spectral band inputs and a chromatic focal shift.

Endomicroscopic optical coherence tomography for cellular resolution imaging of gastrointestinal tracts.

Our ability to detect neoplastic changes in gastrointestinal (GI) tracts is limited by the lack of an endomicroscopic imaging tool that provides cellular-level structural details of GI mucosa over a large tissue area. In this article, we report a fiber-optic-based micro-optical coherence tomography (µOCT) system and demonstrate its capability to acquire cellular-level details of GI tissue through circumferential scanning. The system achieves an axial resolution of 2.48 µm in air and a transverse resolution of 4.8 µm with a depth-of-focus (DOF) of ~150 µm. To mitigate the issue of limited DOF, we used a rigid sheath to maintain a circular lumen and center the distal-end optics. The sensitivity is tested to be 98.8 dB with an illumination power of 15.6 mW on the sample. With fresh swine colon tissues imaged ex vivo, detailed structures such as crypt lumens and goblet cells can be clearly resolved, demonstrating that this fiber-optic µOCT system is capable of visualizing cellular-level morphological features. We also demonstrate that time-lapsed frame averaging and imaging speckle reduction are essential for clearly visualizing cellular-level details. Further development of a clinically viable µOCT endomicroscope is likely to improve the diagnostic outcome of GI cancers.

Polarization management to mitigate misalignment-induced fringe fading in fiber-based optical coherence tomography.

In fiber-based optical coherence tomography (OCT), the interference fringes suffer from the fading effect due to misalignment of the light polarization states between the reference and sample arms, resulting in sensitivity degradation and image intensity variation. We theoretically and experimentally analyzed the relation between the misalignment and the fading coefficient. Assuming that the variation of the light polarization in single-mode fiber (SMF) was a random process, we statistically quantified the fading effect. Furthermore, in OCT configuration based on the Michelson interferometer, we reported an interesting observation that the polarization states of light traveling a round-trip in SMF are not evenly distributed on the Poincare sphere. Based on this observation, we demonstrated the existence of an optimal output polarization state of the reference arm to mitigate the fading effect. We demonstrated that in an optimal setup, the statistical average signal-to-noise ratio could be 3.5 dB higher than a setup without proper polarization management.

Visualizing Micro-anatomical Structures of the Posterior Cornea with Micro-optical Coherence Tomography.

Diagnosis of corneal disease and challenges in corneal transplantation require comprehensive understanding of corneal anatomy, particularly that of the posterior cornea. Micro-optical coherence tomography (µOCT) is a potentially suitable tool to meet this need, owing to its ultrahigh isotropic spatial resolution, high image acquisition rate and depth priority scanning mode. In this study, we explored the ability of µOCT to visualize micro-anatomical structures of the posterior cornea ex vivo and in vivo using small and large animals. µOCT clearly delineated cornea layers and revealed micro-anatomical structures, including not only polygonal endothelial cells, stellate keratocytes, collagen fibres and corneal nerve fibres but also new structures such as the dome-shaped basolateral side of endothelial cells and lattice structures at the interface between endothelium and Descemet's membrane. Based on these observations, a short post-harvest longitudinal study was conducted on rat cornea to test the feasibility of using µOCT to monitor the quality of endothelial cells. This study successfully reveals a series of morphological features and pathological changes in the posterior cornea at the cellular level in situ and in real time with µOCT. These findings enrich knowledge of corneal anatomy and suggest that µOCT may be a promising imaging tool in corneal transplantation.