Optimal operational conditions for supercontinuum-based ultrahigh-resolution endoscopic OCT imaging.

We investigated the optimal operational conditions for utilizing a broadband supercontinuum (SC) source in a portable 800 nm spectral-domain (SD) endoscopic OCT system to enable high resolution, high-sensitivity, and high-speed imaging in vivo. A SC source with a 3-dB bandwidth of ∼246 nm was employed to obtain an axial resolution of ∼2.7 µm (in air) and an optimal detection sensitivity of ∼-107 dB with an imaging speed up to 35 frames/s (at 70 k A-scans/s). The performance of the SC-based SD-OCT endoscopy system was demonstrated by imaging guinea pig esophagus in vivo, achieving image quality comparable to that acquired with a broadband home-built Ti:sapphire laser.

Allergen challenge sensitizes TRPA1 in vagal sensory neurons and afferent C-fiber subtypes in guinea pig esophagus.

Transient receptor potential A1 (TRPA1) is a newly defined cationic ion channel, which selectively expresses in primary sensory afferent nerve, and is essential in mediating inflammatory nociception. Our previous study demonstrated that TRPA1 plays an important role in tissue mast cell activation-induced increase in the excitability of esophageal vagal nodose C fibers. The present study aims to determine whether prolonged antigen exposure in vivo sensitizes TRPA1 in a guinea pig model of eosinophilic esophagitis (EoE). Antigen challenge-induced responses in esophageal mucosa were first assessed by histological stains and Ussing chamber studies. TRPA1 function in vagal sensory neurons was then studied by calcium imaging and by whole cell patch-clamp recordings in 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled esophageal vagal nodose and jugular neurons. Extracellular single-unit recordings were performed in vagal nodose and jugular C-fiber neuron subtypes using ex vivo esophageal-vagal preparations with intact nerve endings in the esophagus. Antigen challenge significantly increased infiltrations of eosinophils and mast cells in the esophagus. TRPA1 agonist allyl isothiocyanate (AITC)-induced calcium influx in nodose and jugular neurons was significantly increased, and current densities in esophageal DiI-labeled nodose and jugular neurons were also significantly increased in antigen-challenged animals. Prolonged antigen challenge decreased esophageal epithelial barrier resistance, which allowed intraesophageal-infused AITC-activating nodose and jugular C fibers at their nerve endings. Collectively, these results demonstrated that prolonged antigen challenge sensitized TRPA1 in esophageal sensory neurons and afferent C fibers. This novel finding will help us to better understand the molecular mechanism underlying esophageal sensory and motor dysfunctions in EoE.

A compact fiber-optic SHG scanning endomicroscope and its application to visualize cervical remodeling during pregnancy.

We report the development of an all-fiber-optic scanning endomicroscope capable of high-resolution second harmonic generation (SHG) imaging of biological tissues and demonstrate its utility for monitoring the remodeling of cervical collagen during gestation in mice. The endomicroscope has an overall 2.0 mm diameter and consists of a single customized double-clad fiber, a compact rapid two-dimensional beam scanner, and a miniature compound objective lens for excitation beam delivery, scanning, focusing, and efficient SHG signal collection. Endomicroscopic SHG images of murine cervical tissue sections at different stages of normal pregnancy reveal progressive, quantifiable changes in cervical collagen morphology with resolution similar to that of bench-top SHG microscopy. SHG endomicroscopic imaging of ex vivo murine and human cervical tissues through intact epithelium has also been performed. Our findings demonstrate the feasibility of SHG endomicroscopy technology for staging normal pregnancy, and suggest its potential application as a minimally invasive tool for clinical assessment of abnormal cervical remodeling associated with preterm birth.

Generic pixel-wise speckle detection in Fourier-domain optical coherence tomography images.

We present a generic phase-domain processing method for detecting speckles in Fourier-domain optical coherence tomography (OCT) images. The physics behind the interferometry is revisited and analytically along with simulation results it indicates that the speckle formation comes with phase distortion to the complex OCT signal. The first and the second derivatives of phase along the imaging depth are then calculated for speckle identification. The phase-domain processing method was applied to images acquired by both spectral-domain OCT and swept-source OCT systems, and the experimental results show that this method enables pixel-wise speckle identification.

Diffractive catheter for ultrahigh-resolution spectral-domain volumetric OCT imaging.

We present a novel design for an endoscopic imaging catheter utilizing diffractive optics for ultrahigh-resolution optical coherence tomography (OCT) imaging at 800 nm. A diffractive microlens was developed to alleviate severe chromatic aberration when a broadband light source was employed at the 800 nm wavelength range. Combined with a home-built fiber rotary joint and a broadband Ti:sapphire laser, the imaging catheter achieved a lateral resolution of 6.2 µm and an axial resolution of 3.0 µm in air. The performance of the catheter was demonstrated by three-dimensional full-circumferential endoscopic OCT imaging of guinea pig esophagus in vivo.

Identification, prioritization, and evaluation of glycoproteins for aggressive prostate cancer using quantitative glycoproteomics and antibody-based assays on tissue specimens.

Prostate cancer is highly heterogeneous in nature; while the majority of cases are clinically insignificant, some cases are lethal. Currently, there are no reliable screening methods for aggressive prostate cancer. Since most established serum and urine biomarkers are glycoproteins secreted or leaked from the diseased tissue, the current study seeks to identify glycoprotein markers specific to aggressive prostate cancer using tissue specimens. With LC-MS/MS glycoproteomic analysis, we identified 350 glycopeptides with 17 being altered in aggressive prostate cancer. ELISA assays were developed/purchased to evaluate four candidates, that is, cartilage oligomeric matrix protein (COMP), periostin, membrane primary amine oxidase (VAP-1), and cathepsin L, in independent tissue sets. In agreement with the proteomic analysis, we found that COMP and periostin expressions were significantly increased in aggressive prostate tumors while VAP-1 expression was significantly decreased in aggressive tumor. In addition, the expression of these proteins in prostate metastases also follows the same pattern observed in the proteomic analysis. This study provides a workflow for biomarker discovery, prioritization, and evaluation of aggressive prostate cancer markers using tissue specimens. Our data suggest that increase in COMP and periostin and decrease in VAP-1 expression in the prostate may be associated with aggressive prostate cancer.

Integrated multimodal endomicroscopy platform for simultaneous en face optical coherence and two-photon fluorescence imaging.

We report an all-fiber-optic scanning, multimodal endomicroscope capable of simultaneous optical coherence tomography (OCT) and two-photon fluorescence (TPF) imaging. Both imaging modalities share the same miniature fiber-optic scanning endomicroscope, which consists of a double-clad fiber with a core operating in single mode at both the OCT (1310 nm) and two-photon excitation (1550 nm) wavelengths, a piezoelectric two-dimensional fiber-optic beam scanner, and a miniature aspherical compound lens suitable for simultaneous acquisition of en face OCT and TPF images. A fiber-optic wavelength division multiplexer was employed in the integrated platform to combine the low coherence OCT light source and the femtosecond two-photon excitation laser into the same optical path. Preliminary imaging results of cell cultures and mouse tissue ex vivo demonstrate the feasibility of simultaneous real-time OCT and TPF imaging in a scanning endomicroscopy setting for the first time.

Forward-viewing resonant fiber-optic scanning endoscope of appropriate scanning speed for 3D OCT imaging.

A forward-viewing resonant fiber-optic endoscope of a scanning speed appropriate for a high-speed Fourier-domain optical coherence tomography (FD-OCT) system was developed to enable real-time, three-dimensional endoscopic OCT imaging. A new method was explored to conveniently tune the scanning frequency of a resonant fiber-optic scanner, by properly selecting the fiber-optic cantilever length, partially changing the mechanical property of the cantilever, and adding a weight to the cantilever tip. Systematic analyses indicated the resonant scanning frequency can be tuned over two orders of magnitude spanning from approximately 10Hz to approximately kHz. Such a flexible scanning frequency range makes it possible to set an appropriate scanning speed of the endoscope to match the different A-scan rates of a variety of FD-OCT systems. A 2.4-mm diameter, 62.5-Hz scanning endoscope appropriate to work with a 40-kHz swept-source OCT (SS-OCT) system was developed and demonstrated for 3D OCT imaging of biological tissues.

Generic real-time uniform K-space sampling method for high-speed swept-source optical coherence tomography.

We developed a universal, real-time uniform K-space sampling (Rt-UKSS) method for high-speed swept-source optical coherence tomography (SS-OCT). An external clock uniform in K-space was generated. The clock was synchronized with the zero-crossing time of an interferometric calibration signal and used as triggers for a high-speed data acquisition system in a point-by-point fashion, hence enabling uniform data sampling in K-space. Different from the numerical calibration algorithm commonly used in an SS-OCT system, the method reported here does not require over-sampling, thus greatly reducing the demand for digitization, data processing and storage speed. The Rt-UKSS method is adaptive and applicable to a generic SS-OCT system of a wide range of A-scan rates without special adjustment. We successfully implemented the Rt-UKSS method in an SS-OCT system based on a Fourier-domain mode-locked laser (FDML) of a 40-kHz scanning rate. Real-time imaging of biological tissues using such a system was demonstrated with a measured axial resolution of 9.3 mum and detection sensitivity greater than 120dB.

Scanning all-fiber-optic endomicroscopy system for 3D nonlinear optical imaging of biological tissues.

An extremely compact all-fiber-optic scanning endomicroscopy system was developed for two-photon fluorescence (TPF) and second harmonic generation (SHG) imaging of biological samples. A conventional double-clad fiber (DCF) was employed in the endomicroscope for single-mode femtosecond pulse delivery, multimode nonlinear optical signals collection and fast two-dimensional scanning. A single photonic bandgap fiber (PBF) with negative group velocity dispersion at two-photon excitation wavelength (i.e. approximately 810 nm) was used for pulse prechirping in replacement of a bulky grating/lens-based pulse stretcher. The combined use of DCF and PBF in the endomicroscopy system made the endomicroscope basically a plug-and-play unit. The excellent imaging ability of the extremely compact all-fiber-optic nonlinear optical endomicroscopy system was demonstrated by SHG imaging of rat tail tendon and depth-resolved TPF imaging of epithelial tissues stained with acridine orange. The preliminary results suggested the promising potential of this extremely compact all-fiber-optic endomicroscopy system for real-time assessment of both epithelial and stromal structures in luminal organs.

AI-Assisted In Situ Detection of Human Glioma Infiltration Using a Novel Computational Method for Optical Coherence Tomography.

PURPOSE: In glioma surgery, it is critical to maximize tumor resection without compromising adjacent noncancerous brain tissue. Optical coherence tomography (OCT) is a noninvasive, label-free, real-time, high-resolution imaging modality that has been explored for glioma infiltration detection. Here, we report a novel artificial intelligence (AI)-assisted method for automated, real-time, in situ detection of glioma infiltration at high spatial resolution.Experimental Design: Volumetric OCT datasets were intraoperatively obtained from resected brain tissue specimens of 21 patients with glioma tumors of different stages and labeled as either noncancerous or glioma-infiltrated on the basis of histopathology evaluation of the tissue specimens (gold standard). Labeled OCT images from 12 patients were used as the training dataset to develop the AI-assisted OCT-based method for automated detection of glioma-infiltrated brain tissue. Unlabeled OCT images from the other 9 patients were used as the validation dataset to quantify the method detection performance. RESULTS: Our method achieved excellent levels of sensitivity (∼100%) and specificity (∼85%) for detecting glioma-infiltrated tissue with high spatial resolution (16 µm laterally) and processing speed (∼100,020 OCT A-lines/second). CONCLUSIONS: Previous methods for OCT-based detection of glioma-infiltrated brain tissue rely on estimating the tissue optical attenuation coefficient from the OCT signal, which requires sacrificing spatial resolution to increase signal quality, and performing systematic calibration procedures using tissue phantoms. By overcoming these major challenges, our AI-assisted method will enable implementing practical OCT-guided surgical tools for continuous, real-time, and accurate intraoperative detection of glioma-infiltrated brain tissue, facilitating maximal glioma resection and superior surgical outcomes for patients with glioma.

Pixel-based speckle adjustment for noise reduction in Fourier-domain OCT images.

Speckle resides in OCT signals and inevitably effects OCT image quality. In this work, we present a novel method for speckle noise reduction in Fourier-domain OCT images, which utilizes the phase information of complex OCT data. In this method, speckle area is pre-delineated pixelwise based on a phase-domain processing method and then adjusted by the results of wavelet shrinkage of the original image. Coefficient shrinkage method such as wavelet or contourlet is applied afterwards for further suppressing the speckle noise. Compared with conventional methods without speckle adjustment, the proposed method demonstrates significant improvement of image quality.

MEMS scanning micromirror for optical coherence tomography.

This paper describes an endoscopic-inspired imaging system employing a micro-electromechanical system (MEMS) micromirror scanner to achieve beam scanning for optical coherence tomography (OCT) imaging. Miniaturization of a scanning mirror using MEMS technology can allow a fully functional imaging probe to be contained in a package sufficiently small for utilization in a working channel of a standard gastroesophageal endoscope. This work employs advanced image processing techniques to enhance the images acquired using the MEMS scanner to correct non-idealities in mirror performance. The experimental results demonstrate the effectiveness of the proposed technique.

Detection of human brain cancer infiltration ex vivo and in vivo using quantitative optical coherence tomography.

More complete brain cancer resection can prolong survival and delay recurrence. However, it is challenging to distinguish cancer from noncancer tissues intraoperatively, especially at the transitional, infiltrative zones. This is especially critical in eloquent regions (for example, speech and motor areas). This study tested the feasibility of label-free, quantitative optical coherence tomography (OCT) for differentiating cancer from noncancer in human brain tissues. Fresh ex vivo human brain tissues were obtained from 32 patients with grade II to IV brain cancer and 5 patients with noncancer brain pathologies. On the basis of volumetric OCT imaging data, pathologically confirmed brain cancer tissues (both high- and low-grade) had significantly lower optical attenuation values at both cancer core and infiltrated zones when compared with noncancer white matter, and OCT achieved high sensitivity and specificity at an attenuation threshold of 5.5 mm(-1) for brain cancer patients. We also used this attenuation threshold to confirm the intraoperative feasibility of performing in vivo OCT-guided surgery using a murine model harboring human brain cancer. Our OCT system was capable of processing and displaying a color-coded optical property map in real time at a rate of 110 to 215 frames per second, or 1.2 to 2.4 s for an 8- to 16-mm(3) tissue volume, thus providing direct visual cues for cancer versus noncancer areas. Our study demonstrates the translational and practical potential of OCT in differentiating cancer from noncancer tissue. Its intraoperative use may facilitate safe and extensive resection of infiltrative brain cancers and consequently lead to improved outcomes when compared with current clinical standards.

Characterizing optical properties of nano contrast agents by using cross-referencing OCT imaging.

We report a cross-referencing method to quickly and accurately characterize the optical properties of nanoparticles including the extinction, scattering, absorption and backscattering cross sections by using an OCT system alone. Among other applications, such a method is particularly useful for developing nanoparticle-based OCT imaging contrast agents. The method involves comparing two depth-dependent OCT intensity signals collected from two samples (with one having and the other not having the nanoparticles), to extract the extinction and backscattering coefficient, from which the absorption coefficient can be further deduced (with the help of the established scattering theories for predicting the ratio of the backscattering to total scattering cross section). The method has been experimentally validated using test nanoparticles and was then applied to characterizing gold nanocages. With the aid of this method, we were able to successfully synthesize scattering dominant gold nanocages for the first time and demonstrated the highest contrast enhancement ever achieved by the gold nanocages (and by any nanoparticles of a similar size and concentration) in an in vivo mouse tumor model. This method also enables quantitative analysis of contrast enhancement and provides a general guideline on choosing the optimal concentration and optical properties for the nanoparticle-based OCT contrast agents.

An all-fiber-optic endoscopy platform for simultaneous OCT and fluorescence imaging.

We present an all-fiber-optically based endoscope platform for simultaneous optical coherence tomography (OCT) and fluorescence imaging. This design entails the use of double-clad fiber (DCF) in the endoscope for delivery of OCT source and fluorescence excitation light while collecting the backscattered OCT signal through the single-mode core and fluorescence emission through the large inner cladding of the DCF. Circumferential beam scanning was performed by rotating a 45° reflector using a miniature DC motor at the distal end of the endoscope. Additionally, a custom DCF coupler and a wavelength division multiplexer (WDM) were utilized to seamlessly integrate both imaging modalities to achieve an entirely fiber-optically based dual-modality imaging system. We demonstrated simultaneous intraluminal 3D OCT and 2D (surface) fluorescence imaging in ex vivo rabbit esophagus using the dual-modal endomicroscopy system. Structural morphologies (provided by OCT) and fluorophore distribution (provided by the fluorescence module) could be clearly visualized, suggesting the potential of the dual-modality system for future in vivo and clinical applications.

Self-starting, self-regulating Fourier domain mode locked fiber laser for OCT imaging.

We present a Fourier domain mode locking (FDML) fiber laser with a feedback loop allowing automatic startup without a priori knowledge of the fundamental drive frequency. The feedback can also regulate the drive frequency making the source robust against environmental variations. A control system samples the energy of the light traversing the FDML cavity and uses a voltage controlled oscillator (VCO) to drive the tunable fiber Fabry-Perot filter in order to maximize that energy. We demonstrate a prototype self-starting, self-regulating FDML operating at 40 kHz with a full width tuning range of 140 nm around 1305 nm and a power output of ~40 mW. The laser starts up with no operator intervention in less than 5 seconds and exhibits improved spectral stability over a conventional FDML source. In OCT applications the source achieved over 120 dB detection sensitivity and an ~8.9-µm axial resolution.

Fiber-optic nonlinear endomicroscopy with focus scanning by using shape memory alloy actuation.

A miniature fiber optic endomicroscope with built-in dynamic focus scanning capability is developed for the first time for 3-D two-photon fluorescence (TPF) imaging of biological samples. Fast 2-D lateral beam scanning is realized by resonantly vibrating a double-clad fiber cantilever with a tubular piezoactuator. Slow axial scanning is achieved by moving the distal end of the imaging probe with an extremely compact electrically driven shape memory alloy (SMA). The 10-mm-long SMA allows 150-µm contractions with a driving voltage varying only from 50 to 100 mV. The response of the SMA contraction with the applied voltage is nonlinear, but repeatable and can be accurately calibrated. Depth-resolved imaging of acriflavine-stained biological tissues and unstained white paper with the endomicroscope is performed, and the results demonstrate the feasibility of 3-D nonlinear optical imaging with the SMA-based scanning fiber-optic endomicroscope.

Scanning fiber-optic nonlinear endomicroscopy with miniature aspherical compound lens and multimode fiber collector.

A flexible scanning fiber-optic endomicroscope using a miniature compound lens and a multimode-fiber (MMF) collector was developed for two-photon fluorescence (TPF) and second-harmonic generation (SHG) imaging. The compound lens consisted of a pair of aspherical lenses and exhibited reduced chromatic aberration compared with gradient-index lenses, thus increasing the TPF/SHG collection efficiency. The introduction of a short MMF collector at the distal end of the double-clad fiber of the endomicroscope further mitigated the adverse influence of chromatic aberration of the lens and enhanced the TPF/SHG collection efficiency. Both ray-tracing simulations and experiments on TPF imaging of fluorescent beads and SHG imaging of rattail tendon demonstrated approximately nine (approximately four) times improved collection efficiency for TPF (SHG) with the new endomicroscope design that utilized a compound lens and an MMF collector.

High-resolution OCT balloon imaging catheter with astigmatism correction.

We report new optics designs for an optical coherence tomography (OCT) balloon imaging catheter to achieve diffraction-limited high resolution at a large working distance and enable the correction of severe astigmatism in the catheter. The designs employed a 1 mm diameter gradient-index lens of a properly chosen pitch number and a glass rod spacer to fully utilize the available NA of the miniature optics. Astigmatism caused by the balloon tubing was analyzed, and a method based on a cylindrical reflector was proposed and demonstrated to compensate the astigmatism. A catheter based on the new designs was successfully developed with a measured diffraction-limited lateral resolution of approximately 21 microm, a working distance of approximately 11-12 mm, and a round-shape beam profile. The performance of the OCT balloon catheter was demonstrated by 3D full-circumferential imaging of a swine esophagus in vivo along with a high-speed, Fourier-domain, mode-locked swept-source OCT system.