Neuronal Representation of Social Information in the Medial Amygdala of Awake Behaving Mice.

The medial amygdala (MeA) plays a critical role in processing species- and sex-specific signals that trigger social and defensive behaviors. However, the principles by which this deep brain structure encodes social information is poorly understood. We used a miniature microscope to image the Ca2+ dynamics of large neural ensembles in awake behaving mice and tracked the responses of MeA neurons over several months. These recordings revealed spatially intermingled subsets of MeA neurons with distinct temporal dynamics. The encoding of social information in the MeA differed between males and females and relied on information from both individual cells and neuronal populations. By performing long-term Ca2+ imaging across different social contexts, we found that sexual experience triggers lasting and sex-specific changes in MeA activity, which, in males, involve signaling by oxytocin. These findings reveal basic principles underlying the brain's representation of social information and its modulation by intrinsic and extrinsic factors.

A HIGH-RESOLUTION MICROENDOSCOPE IMPROVES ESOPHAGEAL CANCER SCREENING AND SURVEILLANCE: IMPLICATIONS FOR UNDERSERVED GLOBAL SETTINGS BASED ON AN INTERNATIONAL, RANDOMIZED CONTROLLED TRIAL.

BACKGROUND AND AIMS: Lugol's chromoendoscopy (LCE)-based detection of esophageal squamous cell neoplasia (ESCN) is limited by low specificity. High-resolution microendoscopy (HRME) was shown to improve specificity and reduce unnecessary biopsies when used by academic endoscopists. In this international, randomized controlled trial, we determined the clinical impact, efficiency, and performance of HRME in true global health contexts with a range of providers. METHODS: Subjects undergoing screening or surveillance for ESCN by expert and novice endoscopists were enrolled in China and the U.S. from diverse clinical settings. Subjects were randomized to LCE (standard-of-care) or LCE+HRME (experimental). Primary outcomes were efficiency and clinical impact of LCE vs. LCE+HRME, using gold-standard, consensus pathology. RESULTS: Among 916 consented subjects, 859 (93.8%) were recruited in China and 36 (3.9%) in the U.S.; 21 (2.3%) were excluded due to incomplete procedure or data. In the screening arm, 217 subjects were randomized to LCE, 204 to LCE+HRME; in the surveillance arm, 236 were randomized to LCE, 238 to LCE+HRME. HRME increased efficiency in screening: diagnostic yield (neoplastic/total biopsies) improved from 20.0% (95% confidence interval [CI] 12.7-29.2%) to 51.7% (95% CI 32.5-70.6%) with 65.2% (95% CI 54.6-74.9%) of biopsies potentially saved and 59.7% (95% CI 47.5-71.1%) of subjects potentially spared any biopsy. Six subjects (0.7%) had neoplasia missed by the endoscopist on HRME (false negatives); of these, 3 were moderate or high-grade dysplasia missed by novices. CONCLUSION: A low-cost microendoscope improves the efficiency and clinical impact of ESCN screening and surveillance when combined with LCE. HRME may spare unnecessary biopsies leading to cost savings in underserved global settings where the disease is prevalent. CLINICALTRIALS: gov, Number NCT02029937.

Versatile and automated workflow for the analysis of oligodendroglial calcium signals.

Although intracellular Ca2+ signals of oligodendroglia, the myelin-forming cells of the central nervous system, regulate vital cellular processes including myelination, few studies on oligodendroglia Ca2+ signal dynamics have been carried out and existing software solutions are not adapted to the analysis of the complex Ca2+ signal characteristics of these cells. Here, we provide a comprehensive solution to analyze oligodendroglia Ca2+ imaging data at the population and single-cell levels. We describe a new analytical pipeline containing two free, open source and cross-platform software programs, Occam and post-prOccam, that enable the fully automated analysis of one- and two-photon Ca2+ imaging datasets from oligodendroglia obtained by either ex vivo or in vivo Ca2+ imaging techniques. Easily configurable, our software solution is optimized to obtain unbiased results from large datasets acquired with different imaging techniques. Compared to other recent software, our solution proved to be fast, low memory demanding and faithful in the analysis of oligodendroglial Ca2+ signals in all tested imaging conditions. Our versatile and accessible Ca2+ imaging data analysis tool will facilitate the elucidation of Ca2+-mediated mechanisms in oligodendroglia. Its configurability should also ensure its suitability with new use cases such as other glial cell types or even cells outside the CNS.

Double-Clad Antiresonant Hollow-Core Fiber and Its Comparison with Other Fibers for Multiphoton Micro-Endoscopy.

Label-free and multiphoton micro-endoscopy can transform clinical histopathology by providing an in situ tool for diagnostic imaging and surgical treatment in diseases such as cancer. Key to a multiphoton imaging-based micro-endoscopic device is the optical fiber, for distortion-free and efficient delivery of ultra-short laser pulses to the sample and effective signal collection. In this work, we study a new hollow-core (air-filled) double-clad anti-resonant fiber (DC-ARF) as a high-performance candidate for multiphoton micro-endoscopy. We compare the fiber characteristics of the DC-ARF with a single-clad anti-resonant fiber (SC-ARF) and a solid core fiber (SCF). In this work, while the DC-ARF and the SC-ARF enable low-loss (<0.2 dBm-1), close to dispersion-free excitation pulse delivery (<10% pulse width increase at 900 nm per 1 m fiber) without any induced non-linearities, the SCF resulted in spectral broadening and pulse-stretching (>2000% of pulse width increase at 900 nm per 1 m fiber). An ideal optical fiber endoscope needs to be several meters long and should enable both excitation and collection through the fiber. Therefore, we performed multiphoton imaging on endoscopy-compatible 1 m and 3 m lengths of fiber in the back-scattered geometry, wherein the signals were collected either directly (non-descanned detection) or through the fiber (descanned detection). Second harmonic images were collected from barium titanate crystals as well as from biological samples (mouse tail tendon). In non-descanned detection conditions, the ARFs outperformed the SCF by up to 10 times in terms of signal-to-noise ratio of images. Significantly, only the DC-ARF, due to its high numerical aperture (NA) of 0.45 and wide-collection bandwidth (>1 µm), could provide images in the de-scanned detection configuration desirable for endoscopy. Thus, our systematic characterization and comparison of different optical fibers under different image collection configurations, confirms and establishes the utility of DC-ARFs for high-performing label-free multiphoton imaging-based micro-endoscopy.

S194-Imaging through scattering media by 3D spatial filtering embedded into micro-endoscope.

BACKGROUND: The main objective is related to the capability of integrating into minimally invasive and ultra-thin disposable micro-endoscopic tool, a modality of realizing high-resolution imaging through scattering medium such as blood while performing medical procedure. In this research we aim for the first time to present a time-multiplexing super-resolving approach exhibiting enhanced focus sensitivity, generated by 3D spatial filtering, for significant contrast increase in images collected through scattering medium. METHOD: Our innovative method of imaging through scattering media provides imaging of only one specific object plane in scattering medium's volume while suppressing the noise coming from all other planes. The method should be assisted with axial scanning to perform imaging of the entire 3D object's volume. In our developed optical system noise suppression is achieved by 3D spatial filtering approach while more than an order of magnitude of suppression is experimentally demonstrated. The sensitivity to defocus and noise suppression is dramatically enhanced by placing an array of micro-lenses combined with pinholes raster positioned between two modules of telecentric lenses. RESULTS: We present our novel conceptual designs for the enhanced signal-to-noise ratio (SNR) when imaging through scattering medium and present preliminary experimental results demonstrating both quality imaging performed on resolution bars target as well as SNR quantified results in which SNR enhancement of more than one order of magnitude was obtained. CONCLUSIONS: In this paper, to the best of our knowledge, we present the first ever design of time-multiplexing-based approach for super-resolved imaging through scattering medium. The approach includes a time-multiplexing optical design significantly increasing the depth of focus sensitivity and after performing axial scanning yielding a significant enhancement of the SNR of the 3D object that is being imaged through the scattering medium. Right after the contrast (the SNR) enhancement we scan the object with the projected array of spots (raster) and map it continuously and with high imaging resolution.

Perspective of fibre-optical microendoscopy with microlenses.

Fibre-optical microendoscopy is based on fibre-optical confocal scanning microscopy, where optical fibres are introduced for delivery of the source and collection of the signal. Fibre-optical microendoscopy has led to innovations in imaging of freely moving animals, long-term imaging, minimally invasive diagnostics, and microsurgery. The lens system in fibre-optical microendoscopy is significant because of the imaging resolution and miniaturisation possibility. State-of-the-art fibre-optical microendoscopy based on various types of lens systems is introduced and compared. The lens system contains an objective lens, a gradient index microlens, and other novel lens systems fabricated by electric arc discharge or two-photon lithography.

Optical fibres are introduced in confocal scanning microscopy, inspiring the development of fibre-optical microendoscopy. Fibre-optical microendoscopy takes advantages in imaging of freely moving animals, long-term imaging, minimally invasive diagnostics, and microsurgery. The lens system helps resolving small details of samples and reducing the size in a fibre-optical microendoscope. Various types of lens systems applied in the fibre-optical microendoscopes are introduced and compared, including an objective lens, a gradient index microlens, and other nanofabricated microlens systems.

Cervical lesion assessment using real-time microendoscopy image analysis in Brazil: The CLARA study.

We conducted a prospective evaluation of the diagnostic performance of high-resolution microendoscopy (HRME) to detect cervical intraepithelial neoplasia (CIN) in women with abnormal screening tests. Study participants underwent colposcopy, HRME and cervical biopsy. The prospective diagnostic performance of HRME using an automated morphologic image analysis algorithm was compared to that of colposcopy using histopathologic detection of CIN as the gold standard. To assess the potential to further improve performance of HRME image analysis, we also conducted a retrospective analysis assessing performance of a multi-task convolutional neural network to segment and classify HRME images. One thousand four hundred eighty-six subjects completed the study; 435 (29%) subjects had CIN Grade 2 or more severe (CIN2+) diagnosis. HRME with morphologic image analysis for detection of CIN Grade 3 or more severe diagnoses (CIN3+) was similarly sensitive (95.6% vs 96.2%, P = .81) and specific (56.6% vs 58.7%, P = .18) as colposcopy. HRME with morphologic image analysis for detection of CIN2+ was slightly less sensitive (91.7% vs 95.6%, P < .01) and specific (59.7% vs 63.4%, P = .02) than colposcopy. Images from 870 subjects were used to train a multi-task convolutional neural network-based algorithm and images from the remaining 616 were used to validate its performance. There were no significant differences in the sensitivity and specificity of HRME with neural network analysis vs colposcopy for detection of CIN2+ or CIN3+. Using a neural network-based algorithm, HRME has comparable sensitivity and specificity to colposcopy for detection of CIN2+. HRME could provide a low-cost, point-of-care alternative to colposcopy and biopsy in the prevention of cervical cancer.

Cervical cancer prevention in El Salvador: A prospective evaluation of screening and triage strategies incorporating high-resolution microendoscopy to detect cervical precancer.

Cervical cancer remains a leading cause of cancer death for women in low- and middle-income countries. The goal of our study was to evaluate screening and triage strategies, including high-resolution microendoscopy (HRME), to detect cervical abnormalities concerning for precancer at the point of care. Women (n = 1824) were enrolled at the Instituto de Cáncer de El Salvador. All underwent screening by both human papillomavirus (HPV) testing using careHPV and visual inspection with acetic acid (VIA). Screen-positives, along with 10% of screen-negatives, were invited to return for a follow-up examination that included triage with VIA, colposcopy and HRME imaging. Biopsies were taken of any abnormalities identified. If no abnormalities were identified, then the worst scoring site by HRME was biopsied. The sensitivities of HPV testing and VIA to screen for cervical intraepithelial neoplasia Grade 2 or more severe diagnoses (CIN2+) were 82.1% and 75% (P = .77), while the specificities were 90.4% and 80.9% (P < .001), respectively. The sensitivities of VIA, colposcopy and HRME as triage tests for CIN2+ were 82.1%, 82.1% and 71.4%, respectively (P ≥ .38). HRME had a significantly higher specificity (66.7%) than VIA (51.9%) (P < .001) and colposcopy (53.3%) (P < .001). When evaluating different theoretical screening and triage strategies, screening with HPV testing followed by triage with HRME would result in more women receiving appropriate care (97%) compared to screening with VIA (75%) or HPV alone (90%). Our findings demonstrate that screening with HPV is superior to VIA, and that triage with HRME imaging increases the specificity of detecting CIN2+ at the point of care in a low-resource setting.

A Two-Photon Microimaging-Microdevice System for Four-Dimensional Imaging of Local Drug Delivery in Tissues.

Advances in the intratumor measurement of drug responses have included a pioneering biomedical microdevice for high throughput drug screening in vivo, which was further advanced by integrating a graded-index lens based two-dimensional fluorescence micro-endoscope to monitor tissue responses in situ across time. While the previous system provided a bulk measurement of both drug delivery and tissue response from a given region of the tumor, it was incapable of visualizing drug distribution and tissue responses in a three-dimensional (3D) way, thus missing the critical relationship between drug concentration and effect. Here we demonstrate a next-generation system that couples multiplexed intratumor drug release with continuous 3D spatial imaging of the tumor microenvironment via the integration of a miniaturized two-photon micro-endoscope. This enables optical sectioning within the live tissue microenvironment to effectively profile the entire tumor region adjacent to the microdevice across time. Using this novel microimaging-microdevice (MI-MD) system, we successfully demonstrated the four-dimensional imaging (3 spatial dimensions plus time) of local drug delivery in tissue phantom and tumors. Future studies include the use of the MI-MD system for monitoring of localized intra-tissue drug release and concurrent measurement of tissue responses in live organisms, with applications to study drug resistance due to nonuniform drug distribution in tumors, or immune cell responses to anti-cancer agents.

Limbostomy: Longitudinal Intravital Microendoscopy in Murine Osteotomies.

Bone healing involves the interplay of immune cells, mesenchymal cells, and vasculature over the time course of regeneration. Approaches to quantify the spatiotemporal aspects of bone healing at cellular resolution during long bone healing do not yet exist. Here, a novel technique termed Limbostomy is presented, which combines intravital microendoscopy with an osteotomy. This design allows a modular combination of an internal fixator plate with a gradient refractive index (GRIN) lens at various depths in the bone marrow and can be combined with a surgical osteotomy procedure. The field of view (FOV) covers a significant area of the fracture gap and allows monitoring cellular processes in vivo. The GRIN lens causes intrinsic optical aberrations which have to be corrected. The optical system was characterized and a postprocessing algorithm was developed. It corrects for wave front aberration-induced image plane deformation and for background and noise signals, enabling us to observe subcellular processes. Exemplarily, we quantitatively and qualitatively analyze angiogenesis in bone regeneration. We make use of a transgenic reporter mouse strain with nucleargreen fluorescent protein and membrane-bound tdTomato under the Cadherin-5 promoter. We observe two phases of vascularization. First, rapid vessel sprouting pervades the FOV within 3-4 days after osteotomy. Second, the vessel network continues to be dynamically remodeled until the end of our observation time, 14 days after surgery. Limbostomy opens a unique set of opportunities and allows further insight on spatiotemporal aspects of bone marrow biology, for example, hematopoiesis, analysis of cellular niches, immunological memory, and vascularization in the bone marrow during health and disease. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

The value of microendoscopy in the diagnosis of cervical precancerous lesions and cervical microinvasive carcinoma.

PURPOSE: Cervical cancer is still one of the main causes of death in females. Conventional diagnostic tools such as colposcopy are still unsatisfactory, so accurate diagnostic tools for cervical diseases are needed. Therefore, the purpose of this study was to perform a clinical study to evaluate the value of microendoscopic imaging systems in the diagnosis of cervical precancerous lesions and cervical microinvasive carcinoma (MIC). METHODS: Totally 106 patients ranging in age from 23 to 67 years were recruited. All patients had abnormal thin-layer cytology (TCT) results (≥ low-grade squamous intraepithelial lesions) and high-risk human papillomavirus (HPV) positivity. Each patient was first subjected to ordinary colposcopy, followed by microendoscopy and biopsy. All results of the colposcopy and microendoscopy images were compared to the histopathological diagnosis. RESULTS: Characteristics of pathological blood vessels were easily distinguished by microendoscopy compared with ordinary colposcopy. The diagnostic agreement rate of microendoscopy with the pathological diagnosis was higher (95.3%) than that of ordinary colposcopy (37.7%) (weighted kappa = 0.863, P < .01). When diagnosing HSIL and more advanced disease, the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of the microendoscopic diagnosis were significantly higher than those of ordinary colposcopy (97.6 and 38.1%), (95.5 and 63.6%), (98.8 and 80.0%), (91.3 and 21.2%) and (97.7 and 43.4%), respectively. CONCLUSION: This study shows that microendoscopy has important value in the diagnosis of cervical lesions which can provide real-time diagnosis in vivo without staining, particularly for lesions that are not sensitive to acetic acid staining.

Micro-endoscopic In Vivo Monitoring in the Blood and Lymphatic Vessels of the Oral Cavity after Radiation Therapy.

Radiotherapy, although used worldwide for the treatment of head, neck, and oral cancers, causes acute complications, including effects on vasculature and immune response due to cellular stress. Thus, the ability to diagnose side-effects and monitor vascular response in real-time during radiotherapy would be highly beneficial for clinical and research applications. In this study, recently-developed fluorescence micro-endoscopic technology provides non-invasive, high-resolution, real-time imaging at the cellular level. Moreover, with the application of high-resolution imaging technologies and micro-endoscopy, which enable improved monitoring of adverse effects in GFP-expressing mouse models, changes in the oral vasculature and lymphatic vessels are quantified in real time for 10 days following a mild localized single fractionation, 10 Gy radiotherapy treatments. Fluorescence micro-endoscopy enables quantification of the cardiovascular recovery and immune response, which shows short-term reduction in mean blood flow velocity, in lymph flow, and in transient immune infiltration even after this mild radiation dose, in addition to long-term reduction in blood vessel capacity. The data provided may serve as a reference for the expected cellular-level physiological, cardiovascular, and immune changes in animal disease models after radiotherapy.

Longitudinal micro-endoscopic monitoring of high-success intramucosal xenografts for mouse models of colorectal cancer.

Colorectal cancer (CRC) is one of the most frequently lethal forms of cancer. Intramucosal injection allows development of better mouse models of CRC, as orthotopic xenografts allow development of adenocarcinoma in the submucosa of the mouse colon wall. In this paper, a method of orthotopic injection is monitored longitudinally using cellular-resolution real-time in vivo fluorescence microendoscopy, following the injection of three different cell lines: 3T3-GFP to confirm immunosuppression and HCT116-RFP cells to model CRC. Adenoma formation is first observable after 7 to 10 days, and by use of 33 G needles a tumor induction rate of greater than 85% is documented. An additional experiment on the injection of rapamycin reveals drug efficacy and localization between 24 and 48 hours, and suggests the promise of real-time cellular-resolution fluorescence micro-endoscopy for developing longitudinal therapy regimes in mural models of CRC.

Differential structured illumination microendoscopy for in vivo imaging of molecular contrast agents.

Fiber optic microendoscopy has shown promise for visualization of molecular contrast agents used to study disease in vivo. However, fiber optic microendoscopes have limited optical sectioning capability, and image contrast is limited by out-of-focus light generated in highly scattering tissue. Optical sectioning techniques have been used in microendoscopes to remove out-of-focus light but reduce imaging speed or rely on bulky optical elements that prevent in vivo imaging. Here, we present differential structured illumination microendoscopy (DSIMe), a fiber optic system that can perform structured illumination in real time for optical sectioning without any opto-mechanical components attached to the distal tip of the fiber bundle. We demonstrate the use of DSIMe during in vivo fluorescence imaging in patients undergoing surgery for cervical adenocarcinoma in situ. Images acquired using DSIMe show greater contrast than standard microendoscopy, improving the ability to detect cellular atypia associated with neoplasia.

Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium.

Agonist-mediated signaling by the endothelium controls virtually all vascular functions. Because of the large diversity of agonists, each with varying concentrations, background noise often obscures individual cellular signals. How the endothelium distinguishes low-level fluctuations from noise and decodes and integrates physiologically relevant information remains unclear. Here, we recorded changes in intracellular Ca(2+) concentrations in response to acetylcholine in areas encompassing hundreds of endothelial cells from inside intact pressurized arteries. Individual cells responded to acetylcholine with a concentration-dependent increase in Ca(2+) signals spanning a single order of magnitude. Interestingly, however, intercellular response variation extended over 3 orders of magnitude of agonist concentration, thus crucially enhancing the collective bandwidth of endothelial responses to agonists. We also show the accuracy of this collective mode of detection is facilitated by spatially restricted clusters of comparably sensitive cells arising from heterogeneous receptor expression. Simultaneous stimulation of clusters triggered Ca(2+) signals that were transmitted to neighboring cells in a manner that scaled with agonist concentration. Thus, the endothelium detects agonists by acting as a distributed sensing system. Specialized clusters of detector cells, analogous to relay nodes in modern communication networks, integrate populationwide inputs, and enable robust noise filtering for efficient high-fidelity signaling.-Wilson, C., Saunter, C. D., Girkin, J. M., McCarron, J. G. Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium.

Local domains of motor cortical activity revealed by fiber-optic calcium recordings in behaving nonhuman primates.

Brain mapping experiments involving electrical microstimulation indicate that the primary motor cortex (M1) directly regulates muscle contraction and thereby controls specific movements. Possibly, M1 contains a small circuit "map" of the body that is formed by discrete local networks that code for specific movements. Alternatively, movements may be controlled by distributed, larger-scale overlapping circuits. Because of technical limitations, it remained unclear how movement-determining circuits are organized in M1. Here we introduce a method that allows the functional mapping of small local neuronal circuits in awake behaving nonhuman primates. For this purpose, we combined optic-fiber-based calcium recordings of neuronal activity and cortical microstimulation. The method requires targeted bulk loading of synthetic calcium indicators (e.g., OGB-1 AM) for the staining of neuronal microdomains. The tip of a thin (200 µm) optical fiber can detect the coherent activity of a small cluster of neurons, but is insensitive to the asynchronous activity of individual cells. By combining such optical recordings with microstimulation at two well-separated sites of M1, we demonstrate that local cortical activity was tightly associated with distinct and stereotypical simple movements. Increasing stimulation intensity increased both the amplitude of the movements and the level of neuronal activity. Importantly, the activity remained local, without invading the recording domain of the second optical fiber. Furthermore, there was clear response specificity at the two recording sites in a trained behavioral task. Thus, the results provide support for movement control in M1 by local neuronal clusters that are organized in discrete cortical domains.

In vivo classification of colorectal neoplasia using high-resolution microendoscopy: Improvement with experience.

BACKGROUND AND AIMS: High-resolution microendoscopy (HRME) is a novel, low-cost "optical biopsy" technology that allows for subcellular imaging. The study aim was to evaluate the learning curve of HRME for the differentiation of neoplastic from non-neoplastic colorectal polyps. METHODS: In a prospective cohort fashion, a total of 162 polyps from 97 patients at a single tertiary care center were imaged by HRME and classified in real time as neoplastic (adenomatous, cancer) or non-neoplastic (normal, hyperplastic, inflammatory). Histopathology was the gold standard for comparison. Diagnostic accuracy was examined at three intervals over time throughout the study; the initial interval included the first 40 polyps, the middle interval included the next 40 polyps examined, and the final interval included the last 82 polyps examined. RESULTS: Sensitivity increased significantly from the initial interval (50%) to the middle interval (94%, P = 0.02) and the last interval (97%, P = 0.01). Similarly, specificity was 69% for the initial interval but increased to 92% (P = 0.07) in the middle interval and 96% (P = 0.02) in the last interval. Overall accuracy was 63% for the initial interval and then improved to 93% (P = 0.003) in the middle interval and 96% (P = 0.0007) in the last interval. CONCLUSIONS: In conclusion, this in vivo study demonstrates that an endoscopist without prior colon HRME experience can achieve greater than 90% accuracy for identifying neoplastic colorectal polyps after 40 polyps imaged. HRME is a promising modality to complement white light endoscopy in differentiating neoplastic from non-neoplastic colorectal polyps.

A Novel Method for In Vivo Imaging of Solitary Lung Nodules Using Navigational Bronchoscopy and Confocal Laser Microendoscopy.

Solitary pulmonary nodules (SPN) have become increasingly prevalent and diagnostic management remains challenging. We demonstrate a novel technique in which probe-based confocal endomicroscopy (pCLE) could be performed to microimage SPN in vivo and in real-time. Two confocal wavelengths (488 and 660 nm with methylene blue (MB)) were used for elastin network and cellular imaging, respectively using pCLE in conjunction with r-EBUS and virtual navigation. In the first case, the 1-mm Alveoflex was used to image a metastatic melanoma in a subcentimetric nodule in the right middle lobe. In the next case, a malignant 2-cm nodule in the posterior segment of the upper lobe was imaged using the smaller 0.6-mm Cholangioflex. Lastly, we present a benign case revealing confocal characteristics of a nodular lipid pneumonitis. This reports for the first time the feasibility and utility of pCLE in vivo microimaging of SPN using either the Alveoflex or Cholangioflex miniprobes in addition to 660 nm/MB imaging.

Comparing high-resolution microscopy techniques for potential intraoperative use in guiding low-grade glioma resections.

BACKGROUND AND OBJECTIVES: Fluorescence image-guided surgery (FIGS), with contrast provided by 5-ALA-induced PpIX, has been shown to enable a higher extent of resection of high-grade gliomas. However, conventional FIGS with low-power microscopy lacks the sensitivity to aid in low-grade glioma (LGG) resection because PpIX signal is weak and sparse in such tissues. Intraoperative high-resolution microscopy of PpIX fluorescence has been proposed as a method to guide LGG resection, where sub-cellular resolution allows for the visualization of sparse and punctate mitochondrial PpIX production in tumor cells. Here, we assess the performance of three potentially portable high-resolution microscopy techniques that may be used for the intraoperative imaging of human LGG tissue samples with PpIX contrast: high-resolution fiber-optic microscopy (HRFM), high-resolution wide-field microscopy (WFM), and dual-axis confocal (DAC) microscopy. MATERIALS AND METHODS: Thick unsectioned human LGG tissue samples (n = 7) with 5-ALA-induced PpIX contrast were imaged using three imaging techniques (HRFM, WFM, DAC). The average signal-to-background ratio (SBR) was then calculated for each imaging modality (5 images per tissue, per modality). RESULTS: HRFM provides the ease of use and portability of a flexible fiber bundle, and is simple and inexpensive to build. However, in most cases (6/7), HRFM is not capable of detecting PpIX signal from LGGs due to high autofluorescence, generated by the fiber bundle under laser illumination at 405 nm, which overwhelms the PpIX signal and impedes its visualization. WFM is a camera-based method possessing high lateral resolution but poor axial resolution, resulting in sub-optimal image contrast. CONCLUSIONS: Consistent successful detection of PpIX signal throughout our human LGG tissue samples (n = 7), with an acceptable image contrast (SBR >2), was only achieved using DAC microscopy, which offers superior image resolution and contrast that is comparable to histology, but requires a laser-scanning mechanism to achieve optical sectioning.

Applications and advancements in the use of high-resolution microendoscopy for detection of gastrointestinal neoplasia.

The high-resolution microendoscope (HRME) is a novel imaging modality that allows real-time epithelial imaging at subcellular resolution. Used in concert with any standard endoscope, this portable, low cost, "optical biopsy" technology has the ability to provide images of cellular morphology during a procedure. This technology has been the subject of a number of studies investigating its use in screening and surveillance of a range of gastrointestinal neoplasias, including esophageal adenocarcinoma, esophageal squamous cell cancer, colorectal neoplasia, and anal neoplasia. These studies have shown that HRME is a modality that consistently provides high specificity, negative predictive value, and accuracy across different diseases. In addition, they have illustrated that HRME users can be relatively easily trained in a short period of time, and that users have demonstrated solid inter-rater reliability. These features make HRME a potential complement to high-definition white-light imaging, narrow-band imaging, and other red flag technologies in facilitating real-time clinical diagnosis, endoscopic therapy, and margin determination. Further clinical validation is needed to determine whether this translates to reduced procedure times, pathology costs, and follow-up procedures. Finally, the HRME has a relatively simple design compared with other similar technologies, making it portable, simple to maintain, and low cost. This may allow the HRME device to function in both advanced care settings as well as in places with less resources and specialized support systems. As a whole, the HRME device has shown good performance along with low cost and portable construction, and its application in different conditions and settings has been promising.