High-speed fiber-optic scanning nonlinear endomicroscopy for imaging neuron dynamicsin vivo.

Fiber-optic-based two-photon fluorescence endomicroscopy is emerging as an enabling technology for in vivo histological imaging of internal organs and functional neuronal imaging on freely-behaving animals. However, high-speed imaging remains challenging due to the expense of miniaturization and lack of suited fast beam scanners. For many applications, a higher imaging speed is highly desired, especially for monitoring functional dynamics such as transient dendritic responses in neuroscience. This Letter reports the development of a fast fiber-optic scanning endo-microscope with an imaging speed higher than 26 frames/s. In vivo neural dynamics imaging with the high-speed endomicroscope was performed on a freely-behaving mouse over the primary motor cortex that expressed GCaMP6m. The results demonstrate its capability of real-time monitoring of transient neuronal dynamics with very fine temporal resolution.

Ultralow-voltage electrothermal MEMS based fiber-optic scanning probe for forward-viewing endoscopic OCT.

We report an ultralow-voltage, electrothermal (ET) micro-electro-mechanical system (MEMS) based probe for forward-viewing endoscopic optical coherence tomography (OCT) imaging. The fully assembled probe has a diameter of 5.5 mm and a length of 55 mm, including the imaging optics and a 40 mm long fiber-optic cantilever attached on a micro-platform of the bimorph ET MEMS actuator. The ET MEMS actuator provides a sufficient mechanical actuation force as well as a large vertical displacement, achieving up to a 3 mm optical scanning range with only a 3 VACp-p drive voltage with a 1.5 VDC offset. The imaging probe was integrated with a swept-source OCT system of a 100 kHz A-scan rate, and its performance was successfully demonstrated with cross-sectional imaging of biological tissues ex vivo and in vivo at a speed up to 200 frames per second.

Broadband rotary joint for high-speed ultrahigh-resolution endoscopic OCT imaging at 800 nm.

We report the development of a broadband rotary joint for high-speed ultrahigh-resolution endoscopic optical coherence tomography (OCT) imaging in the 800 nm spectral range. This rotary joint features a pair of achromatic doublets in order to achieve broadband operation for a 3 dB bandwidth over 150 nm. The measured one-way throughput of the rotary joint is greater than 80%, while the fluctuation of the double-pass coupling efficiency during 360 deg rotation is less than ±5% at a near video-rate speed of 20 revolutions/s (rps). The rotary joint is used in conjunction with a diffractive-optics-based endoscope and 800 nm spectral domain OCT system and achieved an ultrahigh axial resolution of ∼2.4 µm in air. The imaging performance is demonstrated by 3D circumferential imaging of a mouse colon in vivo.

Electrothermal MEMS fiber scanner for optical endomicroscopy.

We report a novel MEMS fiber scanner with an electrothermal silicon microactuator and a directly mounted optical fiber. The microactuator comprises double hot arm and cold arm structures with a linking bridge and an optical fiber is aligned along a silicon fiber groove. The unique feature induces separation of resonant scanning frequencies of a single optical fiber in lateral and vertical directions, which realizes Lissajous scanning during the resonant motion. The footprint dimension of microactuator is 1.28 x 7 x 0.44 mm3. The resonant scanning frequencies of a 20 mm long optical fiber are 239.4 Hz and 218.4 Hz in lateral and vertical directions, respectively. The full scanned area indicates 451 µm x 558 µm under a 16 Vpp pulse train. This novel laser scanner can provide many opportunities for laser scanning endomicroscopic applications.

Electrothermal MEMS parallel plate rotation for single-imager stereoscopic endoscopes.

This work reports electrothermal MEMS parallel plate-rotation (PPR) for a single-imager based stereoscopic endoscope. A thin optical plate was directly connected to an electrothermal MEMS microactuator with bimorph structures of thin silicon and aluminum layers. The fabricated MEMS PPR device precisely rotates an transparent optical plate up to 37° prior to an endoscopic camera and creates the binocular disparities, comparable to those from binocular cameras with a baseline distance over 100 µm. The anaglyph 3D images and disparity maps were successfully achieved by extracting the local binocular disparities from two optical images captured at the relative positions. The physical volume of MEMS PPR is well fit in 3.4 mm x 3.3 mm x 1 mm. This method provides a new direction for compact stereoscopic 3D endoscopic imaging systems.

Lissajous fiber scanning for forward viewing optical endomicroscopy using asymmetric stiffness modulation.

We report a fully packaged and compact forward viewing endomicroscope by using a resonant fiber scanner with two dimensional Lissajous trajectories. The fiber scanner comprises a single mode fiber with additional microstructures mounted inside a piezoelectric tube with quartered electrodes. The mechanical cross-coupling between the transverse axes of a resonant fiber with a circular cross-section was completely eliminated by asymmetrically modulating the stiffness of the fiber cantilever with silicon microstructures and an off-set fiber fragment. The Lissajous fiber scanner was fully packaged as endomicroscopic catheter passing through the accessory channel of a clinical endoscope and combined with spectral domain optical coherence tomography (SD-OCT). Ex-vivo 3D OCT images were successfully reconstructed along Lissajous trajectory. The preview imaging capability of the Lissajous scanning enables rapid 3D imaging with high temporal resolution. This endoscopic catheter provides many opportunities for on-demand and non-invasive optical biopsy inside a gastrointestinal endoscope.

Micromachined tethered silicon oscillator for an endomicroscopic Lissajous fiber scanner.

This work reports micromachined tethered silicon oscillators (MTSOs) for endoscopic Lissajous fiber scanners. An MTSO comprises an offset silicon spring for stiffness modulation of a scanning fiber and additional mass for modulation of resonant scanning frequency in one body. MTSOs were assembled with a resonant fiber scanner and enhanced scanning reliability of the scanner by eliminating mechanical cross coupling. The fiber scanner with MTSOs was fully packaged as an endomicroscopic catheter and coupled with a conventional laparoscope and spectral domain OCT system. The endomicroscope was maneuvered with the integrated laparoscope and in vivo swine tissue OCT imaging was successfully demonstrated during open surgery. This new component serves as an important element inside an endoscopic Lissajous fiber scanner for early cancer detection or on-demand minimum lesional margin decision during noninvasive endoscopic biopsy.

Myeloma progression and urinary gammaglobulin affect the urinary cystatin C to diagnose acute kidney injury in multiple myeloma.

AIMS: Urinary cystatin C has been suggested as a useful biomarker for diagnosis of acute kidney injury (AKI). Multiple myeloma is often complicated by AKI. Therefore, we investigated whether the urinary cystatin C was available for diagnosis of AKI in multiple myeloma. MATERIALS AND METHODS: This study included 39 patients with monoclonal gammopathy of undetermined significance (MGUS) or multiple myeloma. We reviewed the medical records retrospectively and investigated whether urinary γ-globulin and myeloma progression had effects on urinary cystatin C excretion. RESULTS: Spearman's correlation analysis showed that serum ß2-microglobulin and serum cystatin C had a significant positive correlation with the urinary cystatin C excretion (r = 0.513, p = 0.001, r = 0.659, p < 0.001) and FEcystatinC (r = 0.585, p = 0.002, r = 0.711, p < 0.001). The GFRcr also had a significant negative correlation with the urinary cystatin C excretion (r = -0.582, p < 0.001) and FEcystatinC (r = -0.474, p = 0.002). In addition, the urinary γ-globulin had a significant positive correlation with the urinary cystatin C excretion (r = 0.678, p < 0.001) and FEcystatinC (r = 0.731, p < 0.001). Urinary γ-globulin was the most significant factor to influence urinary cystatin C excretion in multiple regression test. CONCLUSION: These results indicate that urinary γ-globulin and myeloma progression can increase the fractional and total excretion of urinary cystatin C. Therefore, it is believed that the urinary cystatin C can be affected by urinary γ-globulin and myeloma progression in the diagnosis of AKI in multiple myeloma. In addition, urinary γ-globulin is believed to be the most significant factor to influence on urinary cystatin C.

Multifunctional Ablative Gastrointestinal Imaging Capsule (MAGIC) for Esophagus Surveillance and Interventions.

Objective and Impact Statement: A clinically viable technology for comprehensive esophagus surveillance and potential treatment is lacking. Here, we report a novel multifunctional ablative gastrointestinal imaging capsule (MAGIC) technology platform to address this clinical need. The MAGIC technology could also facilitate the clinical translation and adoption of the tethered capsule endomicroscopy (TCE) technology. Introduction: Recently developed optical coherence tomography (OCT) TCE technologies have shown a promising potential for surveillance of Barrett's esophagus and esophageal cancer in awake patients without the need for sedation. However, it remains challenging with the current TCE technology for detecting early lesions and clinical adoption due to its suboptimal resolution, imaging contrast, and lack of visual guidance during imaging. Methods: Our technology reported here integrates dual-wavelength OCT imaging (operating at 800 and 1300 nm), an ultracompact endoscope camera, and an ablation laser, aiming to enable comprehensive surveillance, guidance, and potential ablative treatment of the esophagus. Results: The MAGIC has been successfully developed with its multimodality imaging and ablation capabilities demonstrated by imaging swine esophagus ex vivo and in vivo. The 800-nm OCT imaging offers exceptional resolution and contrast for the superficial layers, well suited for detecting subtle changes associated with early neoplasia. The 1300-nm OCT imaging provides deeper penetration, essential for assessing lesion invasion. The built-in miniature camera affords a conventional endoscopic view for assisting capsule deployment and laser ablation. Conclusion: By offering complementary and clinically viable functions in a single device, the reported technology represents an effective solution for endoscopic screening, diagnosis, and potential ablation treatment of the esophagus of a patient in an office setting.

Deep learning-based optical coherence tomography image analysis of human brain cancer.

Real-time intraoperative delineation of cancer and non-cancer brain tissues, especially in the eloquent cortex, is critical for thorough cancer resection, lengthening survival, and improving quality of life. Prior studies have established that thresholding optical attenuation values reveals cancer regions with high sensitivity and specificity. However, threshold of a single value disregards local information important to making more robust predictions. Hence, we propose deep convolutional neural networks (CNNs) trained on labeled OCT images and co-occurrence matrix features extracted from these images to synergize attenuation characteristics and texture features. Specifically, we adapt a deep ensemble model trained on 5,831 examples in a training dataset of 7 patients. We obtain 93.31% sensitivity and 97.04% specificity on a holdout set of 4 patients without the need for beam profile normalization using a reference phantom. The segmentation maps produced by parsing the OCT volume and tiling the outputs of our model are in excellent agreement with attenuation mapping-based methods. Our new approach for this important application has considerable implications for clinical translation.

Forward imaging OCT endoscopic catheter based on MEMS lens scanning.

This Letter reports a fully packaged microelectromechanical system (MEMS) endoscopic catheter for forward imaging optical coherence tomography (OCT). Two-dimensional optical scanning of Lissajous patterns was realized by the orthogonal movement of two commercial aspherical glass lenses laterally mounted on two resonating electrostatic MEMS microstages at low operating voltages. The MEMS lens scanner was integrated on a printed circuit board and packaged with an aluminum housing, a gradient index fiber collimator, and an objective lens. A home-built spectral-domain OCT system with 60 kHz A-line acquisition rate was combined with the endoscopic MEMS catheter. Three-dimensional images of 256×256×995 voxels were directly reconstructed by mapping the A-line datasets along the Lissajous patterns. The endoscopic catheter can provide a new direction for forward endoscopic OCT imaging.

Direct Visualization and Quantitative Imaging of Small Airway Anatomy In Vivo Using Deep Learning Assisted Diffractive OCT.

OBJECTIVE/BACKGROUND: In vivo imaging and quantification of the microstructures of small airways in three dimensions (3D) allows a better understanding and management of airway diseases, such as asthma and chronic obstructive pulmonary disease (COPD). At present, the resolution and contrast of the currently available conventional optical coherence tomography (OCT) imaging technologies operating at 1300 nm remain challenging to directly visualize the fine microstructures of small airways in vivo. METHODS: We developed an ultrahigh-resolution diffractive endoscopic OCT at 800 nm to afford a resolving power of 1.7 µm (in tissue) with an improved contrast and a custom deep residual learning based image segmentation framework to perform accurate and automated 3D quantification of airway anatomy. RESULTS: The 800-nm diffractive OCT enabled the direct delineation of the structural components in the small airway wall in vivo. We further first demonstrated the 3D anatomic quantification of critical tissue compartments of small airways in sheep using the automated segmentation method. CONCLUSION: The deep learning assisted diffractive OCT provides a unique ability to access the small airways, directly visualize and quantify the important tissue compartments, such as airway smooth muscle, in the airway wall in vivo in 3D. SIGNIFICANCE: These pilot results suggest a potential technology for calculating volumetric measurements of small airways in patients in vivo.

Visualization and Validation of The Microstructures in The Airway Wall in vivo Using Diffractive Optical Coherence Tomography.

RATIONALE AND OBJECTIVES: At present, there is no available method to study the in vivo microstructures of the airway wall (epithelium, smooth muscle, adventitia, basement membrane, glands, cartilage). Currently, we rely on ex vivo histologic evaluation of airway biopsies. To overcome this obstacle, we have developed an endoscopic ultrahigh-resolution diffractive optical coherence tomography (OCT) system, operating at a wavelength of 800 nm, to non-invasively study the in vivo microstructures of the airway wall. Prior to human study, validation of diffractive OCT's ability to quantitate airway microstructural components is required. MATERIALS AND METHODS: To validate and demonstrate the accuracy of this OCT system, we used an ovine model to image small airways (∼ 2 mm in diameter). Histologic samples and correlated OCT images were matched. The cross-sectional area of the airway wall, lumen, and other microstructures were measured and compared. RESULTS: A total of 27 sheep were studied from which we identified 39 paired OCT-histology airway images. We found strong correlations between the OCT and the histology measurements of the airway wall area and the microstructural area measurements of the epithelium, basement membrane, airway smooth muscle, glands, cartilage, and adventitia. The correlations ranged from r=0.61 (p<0.001) for the epithelium to r=0.86 (p<0.001) for the adventitia with the correlation between the OCT and the histology measurements for the entire airway wall of r=0.76 (p<0.001). CONCLUSION: Given the high degree of correlation, these data validate the ability to acquire and quantify in vivo microscopic level imaging with this newly developed 800nm ultra-high resolution diffractive OCT system.

Deep-learning two-photon fiberscopy for video-rate brain imaging in freely-behaving mice.

Scanning two-photon (2P) fiberscopes (also termed endomicroscopes) have the potential to transform our understanding of how discrete neural activity patterns result in distinct behaviors, as they are capable of high resolution, sub cellular imaging yet small and light enough to allow free movement of mice. However, their acquisition speed is currently suboptimal, due to opto-mechanical size and weight constraints. Here we demonstrate significant advances in 2P fiberscopy that allow high resolution imaging at high speeds (26 fps) in freely-behaving mice. A high-speed scanner and a down-sampling scheme are developed to boost imaging speed, and a deep learning (DL) algorithm is introduced to recover image quality. For the DL algorithm, a two-stage learning transfer strategy is established to generate proper training datasets for enhancing the quality of in vivo images. Implementation enables video-rate imaging at ~26 fps, representing 10-fold improvement in imaging speed over the previous 2P fiberscopy technology while maintaining a high signal-to-noise ratio and imaging resolution. This DL-assisted 2P fiberscope is capable of imaging the arousal-induced activity changes in populations of layer2/3 pyramidal neurons in the primary motor cortex of freely-behaving mice, providing opportunities to define the neural basis of behavior.

Minimizing OCT quantification error via a surface-tracking imaging probe.

OCT-based quantitative tissue optical properties imaging is a promising technique for intraoperative brain cancer assessment. The attenuation coefficient analysis relies on the depth-dependent OCT intensity profile, thus sensitive to tissue surface positions relative to the imaging beam focus. However, it is almost impossible to maintain a steady tissue surface during intraoperative imaging due to the patient's arterial pulsation and breathing, the operator's motion, and the complex tissue surface geometry of the surgical cavity. In this work, we developed an intraoperative OCT imaging probe with a surface-tracking function to minimize the quantification errors in optical attenuation due to the tissue surface position variations. A compact OCT imaging probe was designed and engineered to have a long working distance of ∼ 41 mm and a large field of view of 4 × 4 mm2 while keeping the probe diameter small (9 mm) to maximize clinical versatility. A piezo-based linear motor was integrated with the imaging probe and controlled based upon real-time feedback of tissue surface position inferred from OCT images. A GPU-assisted parallel processing algorithm was implemented, enabling detection and tracking of tissue surface in real-time and successfully suppressing more than 90% of the typical physiologically induced motion range. The surface-tracking intraoperative OCT imaging probe could maintain a steady beam focus inside the target tissue regardless of the surface geometry or physiological motions and enabled to obtain tissue optical attenuation reliably for assessing brain cancer margins in challenging intraoperative settings.

Label-free imaging of human brain tissue at subcellular resolution for potential rapid intra-operative assessment of glioma surgery.

Background: Frozen section and smear preparation are the current standard for intraoperative histopathology during cancer surgery. However, these methods are time-consuming and subject to limited sampling. Multiphoton microscopy (MPM) is a high-resolution non-destructive imaging technique capable of optical sectioning in real time with subcellular resolution. In this report, we systematically investigated the feasibility and translation potential of MPM for rapid histopathological assessment of label- and processing-free surgical specimens. Methods: We employed a customized MPM platform to capture architectural and cytological features of biological tissues based on two-photon excited NADH and FAD autofluorescence and second harmonic generation from collagen. Infiltrating glioma, an aggressive disease that requires subcellular resolution for definitive characterization during surgery, was chosen as an example for this validation study. MPM images were collected from resected brain specimens of 19 patients and correlated with histopathology. Deep learning was introduced to assist with image feature recognition. Results: MPM robustly captures diagnostic features of glioma including increased cellularity, cellular and nuclear pleomorphism, microvascular proliferation, necrosis, and collagen deposition. Preliminary application of deep learning to MPM images achieves high accuracy in distinguishing gray from white matter and cancer from non-cancer. We also demonstrate the ability to obtain such images from intact brain tissue with a multiphoton endomicroscope for intraoperative application. Conclusion: Multiphoton imaging correlates well with histopathology and is a promising tool for characterization of cancer and delineation of infiltration within seconds during brain surgery.

Micromachined lens microstages for two-dimensional forward optical scanning.

This work presents a novel approach for a miniaturized optical scanning module based on lateral and piston motion of two commercial lenses by MEMS actuation. Two aspheric glass lenses of 1 mm diameter are assembled on two electrostatically actuated microstages moving along perpendicular axes to tilt optical path. The compact integration secures the effective beam aperture of 0.6 mm within the device width of 2 mm. The lens mass provides high-Q motions at low operating voltages of DC 5 V and AC 10 V, i.e., the lateral angle of 4.6 degrees at 277 Hz and the vertical angle of 5.3 degrees at 204 Hz. The device can provide a new direction for miniaturizing laser scanning based endoscopes or handheld projectors.

In vivo assessment of vascular-targeted photodynamic therapy effects on tumor microvasculature using ultrahigh-resolution functional optical coherence tomography.

Vascular-targeted photodynamic therapy (VTP) is an emerging treatment for tumors. The change of tumor vasculatures, including a newly-formed microvascular, in response to VTP, is a key assessment parameter for optimizing the treatment effect. However, an accurate assessment of vasculature, particularly the microvasculature's changes in vivo, remains challenging due to the limited resolution afforded by existing imaging modalities. In this study, we demonstrated the in vivo imaging of VTP effects on an A431 tumor-bearing window chamber model of a mouse with an 800-nm ultrahigh-resolution functional optical coherence tomography (UHR-FOCT). We further quantitatively demonstrated the effects of VTP on the size and density of tumor microvasculature before, during, and after the treatment. Our results suggest the promising potential of UHR-FOCT for assessing the tumor treatment with VTP in vivo and in real time to achieve an optimal outcome.

Ultra-sensitive optical coherence elastography using a high-dynamic-range force loading scheme for cervical rigidity assessment.

An ultra-sensitive, wide-range force loading scheme is proposed for compression optical coherence elastography (OCE) that allows for the quantitative analysis of cervical tissue elasticity ex vivo. We designed a force loading apparatus featuring a water sink for minuscule incremental loading through a volume-controlled water droplet, from which the Young's modulus can be calculated by fitting the stress-strain curve. We validated the performance of the proposed OCE system on homogenous agar phantoms, showing the Young's modulus can be accurately estimated using this scheme. We then measured the Young's modulus of rodent cervical tissues acquired at different gestational ages, showing that the cervical rigidity of rodents was significantly dropped when entering the third trimester of pregnancy.

Throughput-Speed Product Augmentation for Scanning Fiber-Optic Two-Photon Endomicroscopy.

Compactness, among several others, is one unique and very attractive feature of a scanning fiber-optic two-photon endomicroscope. To increase the scanning area and the total number of resolvable pixels (i.e., the imaging throughput), it typically requires a longer cantilever which, however, leads to a much undesired, reduced scanning speed (and thus imaging frame rate). Herein we introduce a new design strategy for a fiber-optic scanning endomicroscope, where the overall numerical aperture (NA) or beam focusing power is distributed over two stages: 1) a mode-field focuser engineered at the tip of a double-clad fiber (DCF) cantilever to pre-amplify the single-mode core NA, and 2) a micro objective of a lower magnification (i.e.,  âˆ¼ 2× in this design) to achieve final tight beam focusing. This new design enables either an ~9-fold increase in imaging area (throughput) or an ~3-fold improvement in imaging frame rate when compared to traditional fiber-optic endomicroscope designs. The performance of an as-designed endomicroscope of an enhanced throughput-speed product was demonstrated by two representative applications: (1) high-resolution imaging of an internal organ (i.e., mouse kidney) in vivo over a large field of view without using any fluorescent contrast agents, and (2) real-time neural imaging by visualizing dendritic calcium dynamics in vivo with sub-second temporal resolution in GCaMP6m-expressing mouse brain. This cascaded NA amplification strategy is universal and can be readily adapted to other types of fiber-optic scanners in compact linear or nonlinear endomicroscopes.