Luminal organ biopsies are critical for disease diagnosis and are obtained using single-bite forceps inserted through the working channel of large endoscopes. Procedures using these endoscopes frequently require patient sedation or anesthesia and may not be feasible for use in pediatric patients. Additionally, forceps-derived biopsies can suffer from difficulty maintaining tissue orientation, crush artifacts, and lack of precise control of biopsy depth. The high cost and risks of anesthesia and sedation have driven the development of smaller endoscopes for unsedated procedures. However, reduced endoscope size limits working-channel dimensions, restricting biopsy forceps to sizes that may yield insufficient or nondiagnostic samples. To address these limitations, we developed an image-guided, depth-controlled, ultrasmall-diameter (1.2-millimeters) cryobiopsy device (μCryoProbe). We optimized the coolant flow profile into the device to enhance tissue freezing, optimizing device-tissue contact time and freezing depth. We tested the device for gastrointestinal biopsy collection in ex vivo preclinical tissues, in an in vivo porcine model, and in sedated human participants. Dimensions and quality of mucosal cryobiopsies from esophagus, stomach, and duodenum were compared with those of forceps-derived biopsies, and it was found that the μCryoProbe device consistently produced high-quality biopsies with optimal tissue orientation and no evidence of crush artifacts. We also demonstrated the ability to capture gastrointestinal biopsies from sedated human participants. By capturing large, well-oriented samples using a small-diameter biopsy tool, this technology has the potential to shift procedures from large to small endoscopes, reducing the need for sedation and improving patient diagnosis through the acquisition of tissue samples with better quality.

Dynamic optical coherence tomography with micrometer resolution (DµOCT) offers enhanced contrast information by evaluating time-dependent signal fluctuations in images of living tissue. DµOCT's use has been limited to imaging excised fresh tissue specimens or 3D cell cultures due to the long observation times required, typically ranging from 1.35 to 25 seconds. To reduce the time needed to obtain DµOCT images, we developed a phase-based algorithm that analyzes intracellular motion by measuring phase changes between adjacent B-scans. This approach significantly reduces imaging time to as low as 40.5 ms while providing a quantitative measure of intracellular motion.

IntroductionBirefringent crystals such as monosodium-urate (MSU) and cholesterol crystals (CC) likely contribute to the progression of coronary artery disease (CAD) due to their potential to exacerbate inflammation through inflammatory cytokine activation. Here, we present cross-polarized micro-optical coherence tomography (CP-µOCT) for visualizing individual birefringent crystals in human coronary arteries.Methods and resultsHuman cadaver coronary arteries with a history of CAD with or without gout were dissected for CP-µOCT imaging. Specimens were processed for histological identification of birefringence under polarization light microscopy (PLM). CP-µOCT visualized needle-crystals that appeared as long projections in orthogonal planes, and PLM confirmed that CP-µOCT-delineated needle-crystals demonstrated negative birefringence. The needle-crystals were dissolved after immersion in uricase (p < 0.05), and thus were MSU. CP-µOCT was three-dimensionally volume-rendered for counting MSU and CCs in 79 regions of interest sized [750 (x) × 500 (y) × 400 (z) µm]. Crystal counts were normalized by the total coronary length utilized. The relationship between CP-µOCT-delineated MSU counts and those seen in corresponding histology, and the difference in coronary MSU amongst gout vs. non-gout patients was analyzed. CP-µOCT-delineated MSU counts were significantly correlated with MSU counted by PLM-based histology (R = 0.98, p < 0.01), and with histology-derived intimal thickening (R = 0.51, p < 0.01). MSU and CCs were both significantly greater in gout patients compared with non-gout patients (p < 0.05).DiscussionThese results demonstrate a significant increase in CP-µOCT-delineated crystals in gout vs. non-gout patients, suggesting that this technology can be used to improve our understanding of crystal-driven coronary pathogenesis.

We have developed a new self-propelled OCT imaging technology called retrograde Tethered Capsule Endomicroscopy (R-TCE) for colonic disease screening. We successfully demonstrated that the R-TCE device can be advanced over 1 meter in 5 swine colons in vivo. R-TCE with balloon pullback imaging enabled full circumferential OCT visualization of 95.94 % ± 0.13% of the colon wall. 3D reconstructed colon OCT images and 3D rendered flythroughs showed that R-TCE is feasible for OCT microscopic imaging of the entire colon in vivo. When translated to humans, this R-TCE technology may provide a less invasive and more efficient alternative to colonoscopy.

We developed OCT-TCE devices with either guidewire or propylene glycol infusion tethers and tested pullback force and tissue damage over different distances of the small intestine in living swine. For all devices, the maximum force was below our safety threshold of 2N across intestinal lengths of 4m or less. At lengths > 4m, the force was > 4N for the infusion tube devices and > 5N for the guidewire devices, and the proximal intestine showed visible damage matching the tether shape. In conclusion, TCE may be safe for jejunal imaging but likely needs further improvement for ileum imaging in humans.

BACKGROUND AND AIMS:Prior coronary optical coherence tomography (OCT)-near infrared auto-fluorescence (NIRAF) imaging data has shown a correlation between high-risk morphological features and NIRAF signal intensity. This study aims to understand the histopathological origins of NIRAF in human cadaver coronary arteries. METHODS:Ex vivo intracoronary OCT-NIRAF imaging was performed on coronary arteries prosected from 23 fresh human cadaver hearts. Arteries with elevated NIRAF were formalin-fixed and paraffin-embedded. Microscopic images of immunostained Glycophorin A (indicating intraplaque hemorrhage) and Sudan Black (indicating ceroid after fixation) stained slides were compared with confocal NIRAF images (ex. 635 nm, em. 655-755 nm) from adjacent unstained slides in each section. Different images from the same section were registered via luminal morphology. Confocal NIRAF-positive 45° sectors were compared to immunohistochemistry and colocalization between NIRAF and intraplaque hemorrhage or ceroid was quantified by Manders' overlap and Dice similarity coefficients. RESULTS:Thirty-one coronary arteries from 14 hearts demonstrated ≥1.5 times higher NIRAF signal than background, and 429 sections were created from them, including 54 sections (12.6%) with high-risk plaques. Within 112 confocal NIRAF-positive 45° sectors, 65 sectors (58.0%) showed both Glycophorin A-positive and Sudan Black-positive, while 7 sectors (6.3%) and 40 sectors (33.6%) only showed Glycophorin A-positive or Sudan black-positive, respectively. A two-tailed McNemar's test showed that Sudan Black more closely corresponded to confocal NIRAF than Glycophorin A (p < 1.0 × 10-6). NIRAF was also found to spatially associate with both Glycophorin A and Sudan Black, with stronger colocalization between Sudan Black and NIRAF (Manders: 0.19 ± 0.15 vs. 0.13 ± 0.14, p < 0.005; Dice: 0.072 ± 0.096 vs. 0.060 ± 0.090, p < 0.01). CONCLUSIONS:As ceroid associates with oxidative stress and intraplaque hemorrhage is implicated in rapid lesion progression, these results suggest that NIRAF provides additional, complementary information to morphologic imaging that may aid in identifying high-risk coronary plaques via translatable intracoronary OCT-NIRAF imaging.

Eosiophilic esophagitis (EoE) is an inflammatory disease of the esophagus, with long-term EoE causing fibrotic and hypertrophic restructuring of the sub-epithelial wall. We have developed a polarization-sensitive micro-optical coherence tomography (PS-µOCT) imaging device for its characterization. This device was used to quantify collagen at 15 sites on each of 5 swine esophagi, with results compared to histology. A linear mixed model with random intercept showed significant agreement between OCT and histology (slope = 0.41, 95% CI [0.22, 0.60], t(67) = 4.30, p = 5.8 x 10-5). This validates our technology and will allow longitudinal assessment of patient response to drug and diet, without endoscopic biopsy.

We report the use of our multimodal near-infrared fluorescence (NIRF) and OCT imaging system and catheter to perform the first imaging of LUM015 inflammatory activity in rabbit models of atherosclerosis in vivo. Using co-injection and multi-channel intravascular NIRF-OCT, we compared LUM015 (6.2 mg/kg) and preclinical ProSense (VM110, 3.5 mg/kg) fluorescence in the same subject. We found that co-registered fluorescence carpet maps were remarkably similar with a PCC of 0.51 and a Mander’s overlap coefficient of 0.79. Results suggest that LUM015 will be a viable clinical option for intracoronary imaging of plaque inflammatory activity in patients.

A screening test for early detection of pancreatic cancer (PC) is a critical unmet need as PC is usually detected late when mortality is unavoidable. Pancreatic fluid (PF), excreted to the duodenum by the Ampulla of Vater (AoV), offers a promising sample for early stage pancreatic cancer screening as it is the richest source of PC bioanalytes. The successful identification of the AoV is critical to develop a minimally invasive and inexpensive capsule-based PC screening test. With our recently developed tether capsule endomicroscopy (TCE) technique, we imaged 27 subjects and analyzed 353 duodenal OCT-TCE datasets. Using relative positions of the major and minor ampulla, and influx of bile into the duodenum, we distinguished the major from the minor. At least one ampulla was identified in 100%, major ampulla identified in 85%, and minor ampulla identified in 67% of all subjects. The measured mean max. diameter of the major ampulla was 6.49 ± 2.23 mm, and 6.09 ± 2.05 mm for the minor.

Micro-optical coherence tomography (µOCT) improves the spatial resolution of in vivo OCT imaging by utilizing sophisticated focusing schemes and broadband illumination. This study explores the safety of coronary and trachea tissue exposure to µOCT illumination. © 2021 The Authors

Introduction: Diseases such as celiac disease, environmental enteric dysfunction, infectious gastroenteritis, type II diabetes and inflammatory bowel disease are associated with increased gut permeability. Dual sugar absorption tests, such as the lactulose to rhamnose ratio (L:R) test, are the current standard for measuring gut permeability. Although easy to administer in adults, the L:R test has a number of drawbacks. These include an inability to assess for spatial heterogeneity in gut permeability that may distinguish different disease severity or pathology, additional sample collection for immunoassays, and challenges in carrying out the test in certain populations such as infants and small children. Here, we demonstrate a minimally invasive probe for real-time localized gut permeability evaluation through gut potential difference (GPD) measurement. Materials and Methods: The probe has an outer diameter of 1.2 mm diameter and can be deployed in the gut of unsedated subjects via a transnasal introduction tube (TNIT) that is akin to an intestinal feeding tube. The GPD probe consists of an Ag/AgCl electrode, an optical probe and a perfusion channel all housed within a transparent sheath. Lactated Ringer’s (LR) solution is pumped through the perfusion channel to provide ionic contact between the electrodes and the gut lining. The optical probe captures non-scanning (M-mode) OCT images to confirm electrode contact with the gut lining. A separate skin patch probe is placed over an abraded skin area to provide reference for the GPD measurements. Swine studies were conducted to validate the GPD probe. GPD in the duodenum was modulated by perfusing 45 ml of 45 mM glucose. Results: GPD values of −13.1 ± 2.8 mV were measured in the duodenum across four swine studies. The change in GPD in the duodenum with the addition of glucose was −10.5 ± 2.4 mV (p < 0.001). M-mode OCT images provided electrode-tissue contact information, which was vital in ascertaining the probe’s proximity to the gut mucosa. Conclusion: We developed and demonstrated a minimally invasive method for investigating gastrointestinal permeability consisting of an image guided GPD probe that can be used in unsedated subjects.

OCT tethered capsule endomicroscopy (TCE) is an emerging noninvasive diagnostic imaging technology for gastrointestinal (GI) tract disorders. OCT measures tissue reflectivity that provides morphologic image contrast, and thus is incapable of ascertaining molecular information that can be useful for improving diagnostic accuracy. Here, we introduce an extension to OCT TCE that includes a fluorescence (FL) imaging channel for attaining complementary, co-registered molecular contrast. We present the development of an OCT-FL TCE capsule and a portable, plug-and-play OCT-FL imaging system. The technology is validated in phantom experiments and feasibility is demonstrated in a methylene blue (MB)-stained swine esophageal injury model, ex vivo and in vivo.

Celiac disease (CD) is an autoimmune disease that damages the small intestine's villi upon gluten ingestion. Intestinal biopsy via esophagogastroduodenoscopy is the current diagnostic gold standard for CD, but this procedure requires sedation and suffers from sampling error. Here, we conducted a clinical study to test whether image biomarkers derived from duodenal OCT tethered capsule endomicroscopy (TCE) can be used to diagnose CD. Results showed a statistically significant difference in OCT image metrics (villus height & width, contrast, and homogeneity with p<0.0001) among active CD, inactive CD and healthy subjects, demonstrating the potential of TCE for the diagnosis of CD.

Objective: Near-infrared autofluorescence (NIRAF) of atherosclerosis associates with intraplaque hemorrhage and is detectable in living patients with coronary artery disease. However, further mechanisms underlying NIRAF generation have not been fully characterized. Here, we investigated the role of lipids and oxidative stress in NIRAF generation in atherosclerosis and in vitro in human macrophages. Approach and Results: In N=15 human carotid endarterectomy specimens, we investigated the spatial distribution of lipid, intraplaque hemorrhage, and NIRAF (ex/em 630/650 nm). Plaque NIRAF associated with both Sudan black-positive lipids (r=0.53, P=0.023) and GPA (glycophorin A)-positive intraplaque hemorrhage (r=0.48, P=0.043). Plaque NIRAF also localized with lipid and specifically insoluble lipid (ceroid) and iron. Intriguingly, some NIRAF-positive areas were Sudan black-positive but GPA-negative. Studies on human macrophages investigated further the role of lipids in NIRAF generation. OxLDL (Oxidized low-density lipoprotein) and hemoglobin, but not LDL, generated NIRAF in both THP-1 cells and monocyte-derived macrophages. In oxLDL-treated THP-1 cells, higher NIRAF, lipid peroxidation products, and intracellular oxidative stress markers evolved (P<0.001 versus LDL). The antioxidants alpha-tocopherol and N-acetylcysteine suppressed NIRAF generation and oxidative stress. Conclusions: In human atherosclerosis and human macrophages in vitro, NIRAF colocalizes with lipid and specifically insoluble lipid or ceroid. In vitro studies further show that oxidized LDL generates NIRAF, oxidative stress, and lipid peroxidation products. These results demonstrate a new pathway for NIRAF generation through oxidized lipid-driven oxidative stress and support ceroid as a source of NIRAF in human atherosclerosis. These findings may inform future clinical intracoronary NIRAF imaging studies of patients with coronary artery disease.

There is significant histopathological and clinical evidence that near-infrared auto-fluorescence (NIRAF) complements optical coherence tomography (OCT) for detecting high-risk coronary plaque. Here, we determined the accuracy of an OCT-NIRAF imaging system and catheter for detecting NIRAF in human coronary lesions. OCT-NIRAF pullback imaging was performed on human cadaver coronary arteries (n=33 from 14 patients) during PBS perfusion via a fully integrated OCT-NIRAF imaging system and catheter (NIRAF ex. 633 nm, 1 mW power; em. 660-740nm). Confocal NIRAF images were acquired from corresponding unstained formalin-fixed paraffin-embedded sections (Olympus FLUOVIEW FV1000; ex. 635 nm; em. 655-755nm). OCT-NIRAF and confocal NIRAF images were registered using known pullback speed, anatomical landmarks, and fiducial features (e.g., calcification), and spatially overlapped by affine transformation of the confocal NIRAF images. Each image was split into 8, 45º-sectors, emanating from the catheter location. Each 45°-sector was determined to be positive if <5% of the intima contained confocal NIRAF, and if <5% of 45°-arc (2.25°) of the catheter-based NIRAF signal was above the system’s detection limit. A total of 1896 45°-sectors from 291 distinct coronary locations were analyzed using confocal NIRAF as the gold standard. Considering superficial confocal NIRAF foci within 0.5 mm from the luminal surface, sensitivity and specificity were 90.0% (95%CI: 69.8- 100.0%) and 90.2% (95%CI: 88.8-91.7%), respectively. Within 0.5 mm to 1.0 mm depth from the luminal surface, the sensitivity was 36.4% (95%CI: 15.0-57.8%) and specificity was 90.1% (95%CI: 88.6-91.5%). These results indicate that the OCT-NIRAF system/catheter’s ability to detect NIRAF is depth dependent and accurate in plaque regions (within 0.5 mm from the luminal surface) that are most responsible for precipitating coronary events.

Living cells exhibit active intracellular molecular motion that reflect their functional states. Traditional microscopy techniques that solely capture high resolution static images of cells misses out on the opportunity to tap into the wealth of information provided by complex intracellular activity. We recently developed dynamic micro-OCT (DμOCT), an extension of μOCT that achieves near-isotropic sub-cellular resolution in all three dimensions (2 µm lateral x 1 µm axial). DμOCT substantially enhanced the contrast of cells and organelles while revealing stratified, depth-dependent dynamics in the epithelial layers. In this work, we have expanded the application DμOCT to encompass imaging of human skin in vivo, and detection of pharmacologic-induced changes in human melanoma spheroids and murine tissues.