MURIN: Multimodal Retinal Imaging and Navigated-laser-delivery for dynamic and longitudinal tracking of photodamage in murine models

IntroductionLaser-induced photodamage is a robust method for investigating retinal pathologies in small animals. However, aiming of the photocoagulation laser is often limited by manual alignment and lacks real-time feedback on lesion location and severity. Here, we demonstrate MURIN: MUltimodal Retinal Imaging and Navigated-laser-delivery, a multimodality OCT and SLO ophthalmic imaging system with an image-guided scanning laser lesioning module optimized for the murine retina. The proposed system enables targeting of focal and extended area lesions under OCT guidance to benefit visualization of photodamage response and the precision and repeatability of laser lesion models of retinal injury.MethodsMURIN optics were optimized for simultaneous near-infrared and visible wavelength imaging/laser lesioning. Custom LabView control software was developed to steer the photocoagulation laser and automatically deliver laser pulses to targets-of-interest. In vivo retinal imaging was performed in transgenic Müller glia-tdTomato reporter mice (Rlbp1:CreER; Rosaai14, 5 animals, 10 eyes) and microglia-GFP/Müller glia-tdTomato reporter mice (Cx3cr1GFP; Rlbp1:CreER; Rosaai14, 9 animals, 15 eyes) to visualize cellular changes in the retina after laser lesion delivery.ResultsReal-time MURIN imaging concurrent with laser lesioning allowed us to visualize lesion formation dynamics and any corresponding changes in retinal morphology. We observe increasing fluorescence photoconversion on SLO and scattering contrast on OCT. Significant morphological changes are visible on MURIN after high-severity photodamage. OCT cross-sections show the spatial extent of the lesions contract over time from diffusion areas of increased scattering to granular scatterers and corresponding SLO images show a radial pattern surrounding severe focal lesions, which may be a result of a change in Müller cell shape or orientation in response to injury. The inner plexiform layer is distorted and increased RPE thickness and scattering are observed, all of which are confirmed on corresponding hematoxylin and eosin (H&E) histology and differential interference contrast (DIC) microscopy.DiscussionMURIN as a unique imaging platform that enables combined SLO and OCT imaging with an integrated image-guided laser lesioning module. This technology has clear benefits over existing multimodal imaging and laser lesioning systems by enabling simultaneous multimodal imaging, independent and precise control of Iridex laser pulse parameters and patterns, and real-time OCT and SLO visualization of lesion formation

Piezoelectric-transducer-based miniature catheter for ultrahigh-speed endoscopic optical coherence tomography

We developed a piezoelectric-transducer- (PZT) based miniature catheter with an outer diameter of 3.5 mm for ultrahigh-speed endoscopic optical coherence tomography (OCT). A miniaturized PZT bender actuates a fiber and the beam is scanned through a GRIN lens and micro-prism to provide high-speed, side-viewing capability. The probe optics can be pulled back over a long distance to acquire three-dimensional (3D) data sets covering a large area. Imaging is performed with 11 μm axial resolution in air (8 μm in tissue) and 20 μm transverse resolution, at 960 frames per second with a Fourier domain mode-locked laser operating at 480 kHz axial scan rate. Using a high-speed data acquisition system, endoscopic OCT imaging of the rabbit esophagus and colon in vivo and human colon specimens ex vivo is demonstrated

Ultrahigh speed endoscopic optical coherence tomography using micromotor imaging catheter and VCSEL technology

We developed a micromotor based miniature catheter with an outer diameter of 3.2 mm for ultrahigh speed endoscopic swept source optical coherence tomography (OCT) using a vertical cavity surface-emitting laser (VCSEL) at a 1 MHz axial scan rate. The micromotor can rotate a micro-prism at several hundred frames per second with less than 5 V drive voltage to provide fast and stable scanning, which is not sensitive to the bending of the catheter. The side-viewing probe can be pulled back to acquire a three-dimensional (3D) data set covering a large area on the specimen. The VCSEL provides a high axial scan rate to support dense sampling under high frame rate operation. Using a high speed data acquisition system, in vivo 3D-OCT imaging in the rabbit GI tract and ex vivo imaging of a human colon specimen with 8 μm axial resolution, 8 μm lateral resolution and 1.2 mm depth range in tissue at a frame rate of 400 fps was demonstrated

Assessment of breast pathologies using nonlinear microscopy

Rapid intraoperative assessment of breast excision specimens is clinically important because up to 40% of patients undergoing breast-conserving cancer surgery require reexcision for positive or close margins. We demonstrate nonlinear microscopy (NLM) for the assessment of benign and malignant breast pathologies in fresh surgical specimens. A total of 179 specimens from 50 patients was imaged with NLM using rapid extrinsic nuclear staining with acridine orange and intrinsic second harmonic contrast generation from collagen. Imaging was performed on fresh, intact specimens without the need for fixation, embedding, and sectioning required for conventional histopathology. A visualization method to aid pathological interpretation is presented that maps NLM contrast from two-photon fluorescence and second harmonic signals to features closely resembling histopathology using hematoxylin and eosin staining. Mosaicking is used to overcome trade-offs between resolution and field of view, enabling imaging of subcellular features over square-centimeter specimens. After NLM examination, specimens were processed for standard paraffin-embedded histology using a protocol that coregistered histological sections to NLM images for paired assessment. Blinded NLM reading by three pathologists achieved 95.4% sensitivity and 93.3% specificity, compared with paraffin-embedded histology, for identifying invasive cancer and ductal carcinoma in situ versus benign breast tissue. Interobserver agreement was κ = 0.88 for NLM and κ = 0.89 for histology. These results show that NLM achieves high diagnostic accuracy, can be rapidly performed on unfixed specimens, and is a promising method for intraoperative margin assessment.National Institutes of Health (U.S.) (Grant R01-CA178636-01)National Institutes of Health (U.S.) (Grant R01-CA75289-16)United States. Air Force Office of Scientific Research (Grant FA9550-10-1-0551)United States. Air Force Office of Scientific Research (Grant FA9550-12-1-0499)National Institutes of Health (U.S.) (National Research Service Award Postdoctoral Fellowship F32-CA165484

Characterization of buried glands before and after radiofrequency ablation by using 3-dimensional optical coherence tomography (with videos)

Background Radiofrequency ablation (RFA) is an endoscopic technique used to eradicate Barrett's esophagus (BE). However, such ablation can commonly lead to neosquamous epithelium overlying residual BE glands not visible by conventional endoscopy and may evade detection on random biopsy samples. Objective To demonstrate the capability of endoscopic 3-dimensional optical coherence tomography (3D-OCT) for the identification and characterization of buried glands before and after RFA therapy. Design Cross-sectional study. Setting Single teaching hospital. Patients Twenty-six male and 1 female white patients with BE undergoing RFA treatment. Interventions 3D-OCT was performed at the gastroesophageal junction in 18 patients before attaining complete eradication of intestinal metaplasia (pre–CE-IM group) and in 16 patients after CE-IM (post–CE-IM group). Main Outcome Measurements Prevalence, size, and location of buried glands relative to the squamocolumnar junction. Results 3D-OCT provided an approximately 30 to 60 times larger field of view compared with jumbo and standard biopsy and sufficient imaging depth for detecting buried glands. Based on 3D-OCT results, buried glands were found in 72% of patients (13/18) in the pre–CE-IM group and 63% of patients (10/16) in the post–CE-IM group. The number (mean [standard deviation]) of buried glands per patient in the post–CE-IM group (7.1 [9.3]) was significantly lower compared with the pre–CE-IM group (34.4 [44.6]; P = .02). The buried gland size (P = .69) and distribution (P = .54) were not significantly different before and after CE-IM. Limitations A single-center, cross-sectional study comparing patients at different time points in treatment. Lack of 1-to-1 coregistered histology for all OCT data sets obtained in vivo. Conclusion Buried glands were frequently detected with 3D-OCT near the gastroesophageal junction before and after radiofrequency ablation.National Institutes of Health (U.S.) (Grant R01-CA75289-15)National Institutes of Health (U.S.) (Grant R44CA101067-06)National Institutes of Health (U.S.) (Grant R01-HL095717-03)National Institutes of Health (U.S.) (Grant R01-NS057476-05)National Institutes of Health (U.S.) (Grant K99-EB010071-01A1)United States. Air Force Office of Scientific Research (Contract FA9550-10-1-0063)United States. Air Force Office of Scientific Research. Medical Free Electron Laser Program (Contract FA9550-10-1-0551)Center for Integration of Medicine and Innovative Technolog

Structural markers observed with endoscopic 3-dimensional optical coherence tomography correlating with Barrett's esophagus radiofrequency ablation treatment response

Background Radiofrequency ablation (RFA) is effective for treating Barrett's esophagus (BE) but often involves multiple endoscopy sessions over several months to achieve complete response. Objective Identify structural markers that correlate with treatment response by using 3-dimensional (3-D) optical coherence tomography (OCT; 3-D OCT). Design Cross-sectional. Setting Single teaching hospital. Patients Thirty-three patients, 32 male and 1 female, with short-segment (<3 cm) BE undergoing RFA treatment. Intervention Patients were treated with focal RFA, and 3-D OCT was performed at the gastroesophageal junction before and immediately after the RFA treatment. Patients were re-examined with standard endoscopy 6 to 8 weeks later and had biopsies to rule out BE if not visibly evident. Main Outcome Measurements The thickness of BE epithelium before RFA and the presence of residual gland-like structures immediately after RFA were determined by using 3-D OCT. The presence of BE at follow-up was assessed endoscopically. Results BE mucosa was significantly thinner in patients who achieved complete eradication of intestinal metaplasia than in patients who did not achieve complete eradication of intestinal metaplasia at follow-up (257 ± 60 μm vs 403 ± 86 μm; P < .0001). A threshold thickness of 333 μm derived from receiver operating characteristic curves corresponded to a 92.3% sensitivity, 85% specificity, and 87.9% accuracy in predicting the presence of BE at follow-up. The presence of OCT-visible glands immediately after RFA also correlated with the presence of residual BE at follow-up (83.3% sensitivity, 95% specificity, 90.6% accuracy). Limitations Single center, cross-sectional study in which only patients with short-segment BE were examined. Conclusion Three-dimensional OCT assessment of BE thickness and residual glands during RFA sessions correlated with treatment response. Three-dimensional OCT may predict responses to RFA or aid in making real-time RFA retreatment decisions in the future.Center for Integration of Medicine and Innovative Technology (Medical Engineering Fellowship)United States. Dept. of Veterans Affairs. Boston Healthcare SystemNational Institutes of Health (U.S.) (Grant R01-CA75289-15)National Institutes of Health (U.S.) (Grant R44CA101067-06)National Institutes of Health (U.S.) (Grant K99-EB010071-01A1)United States. Air Force Office of Scientific Research (Grant FA9550-10-1-0063)United States. Air Force Office of Scientific Research. Medical Free Electron Laser Program (Grant FA9550-10-1-0551

The Feasibility of Spectral-Domain Optical Coherence Tomography Grading of Anterior Chamber Inflammation in a Rabbit Model of Anterior Uveitis

PURPOSE. To determine the feasibility and accuracy of spectral-domain optical coherence tomography (SD-OCT) based grading of anterior chamber cell, using aqueous sampling as a standard, in a rabbit model of anterior uveitis. METHODS. Adult Dutch-belted rabbits were preimmunized with M. tuberculosis (Tb) H37RA antigen, 1 week prior to induction of anterior uveitis with an intracameral injection of Tb antigen. The anterior chamber was imaged with SD-OCT, followed by a slit lamp examination. Two independent, trained graders recorded their estimate of anterior chamber cell count using the Standardization of Uveitis Nomenclature (SUN) scores for each eye prior to performing an anterior chamber tap to determine the aqueous cell density using a hemocytometer. Using the aqueous cell density as a standard, correlation with SD-OCT counts were compared to those with SUN scores. RESULTS. Overall, SD-OCT correlated well with the hemocytometer counts (Spearman coefficient ¼ 0.53, P &lt; 0.001) compared with SUN grading and hemocytometer counts (Spearman coefficient ¼ 0.02, P ¼ 0.88). The correlation improved to 0.65 (P &lt; 0.001) when we excluded eyes with corneal thickness ‡ 470 lm. Eyes with corneal thickness ‡ 470 lm exhibited the greatest degree of ocular inflammation and corneal opacity. CONCLUSIONS. In our rabbit model, SD-OCT grading of anterior chamber cell correlated significantly better with aqueous cell counts, compared to traditional slit lamp grading. Spectral-domain optical coherence tomography grading of anterior chamber cell may be a good alternative to SUN grading. Although SUN grading remains the clinical gold standard, alternative quantitative methods to assess ocular inflammation could provide insight into disease mechanism and aid in measuring treatment response

Ultrahigh speed endoscopic swept source optical coherence tomography using a VCSEL light source and micromotor catheter

We developed an ultrahigh speed endoscopic swept source optical coherence tomography (OCT) system for clinical gastroenterology using a vertical-cavity surface-emitting laser (VCSEL) and micromotor based imaging catheter, which provided an imaging speed of 600 kHz axial scan rate and 8 μm axial resolution in tissue. The micromotor catheter was 3.2 mm in diameter and could be introduced through the 3.7 mm accessory port of an endoscope. Imaging was performed at 400 frames per second with an 8 μm spot size using a pullback to generate volumetric data over 16 mm with a pixel spacing of 5 μm in the longitudinal direction. Three-dimensional OCT (3D-OCT) imaging was performed in patients with a cross section of pathologies undergoing standard upper and lower endoscopy at the Veterans Affairs Boston Healthcare System (VABHS). Patients with Barrett’s esophagus, dysplasia, and inflammatory bowel disease were imaged. The use of distally actuated imaging catheters allowed OCT imaging with more flexibility such as volumetric imaging in the terminal ileum and the assessment of the hiatal hernia using retroflex imaging. The high rotational stability of the micromotor enabled 3D volumetric imaging with micron scale volumetric accuracy for both en face and cross-sectional imaging. The ability to perform 3D OCT imaging in the GI tract with microscopic accuracy should enable a wide range of studies to investigate the ability of OCT to detect pathology as well as assess treatment response.National Institutes of Health (U.S.) (R44EY022864-01)National Institutes of Health (U.S.) (R01-CA75289-17)National Institutes of Health (U.S.) (R44-CA101067-06)National Institutes of Health (U.S.) ( R01-EY011289-27)National Institutes of Health (U.S.) (R01-HL095717-04)National Institutes of Health (U.S.) (R01-NS057476-05)United States. Air Force Office of Scientific Research (FA9550-10-1-0063)United States. Air Force Office of Scientific Research. Medical Free Electron Laser Program (FA9550-10-1-0551)German Research Foundation (DFG-GSC80-SAOT)German Research Foundation (DFG-HO-1791/11-1)Center for Integration of Medicine and Innovative Technolog