Optical Biopsies Using Probe-Based Confocal Laser Endomicroscopy for Autoimmune Pulmonary Alveolar Proteinosis.

INTRODUCTION: Increasing numbers of cases of mild asymptomatic pulmonary alveolar proteinosis (PAP) are being reported with the recent increase in chest computed tomography (CT). Bronchoscopic diagnosis of mild PAP is challenging because of the patchy distribution of lesions, which makes it difficult to obtain sufficient biopsy samples. Additionally, the pathological findings of mild PAP, particularly those that differ from severe PAP, have not been fully elucidated. This study aimed to clarify the pathological findings of mild PAP and the usefulness of optical biopsy using probe-based confocal laser endomicroscopy (pCLE). METHODS: We performed bronchoscopic optical biopsy using pCLE and tissue biopsy in 5 consecutive patients with PAP (three with mild PAP and two with severe PAP). We compared the pCLE images of mild PAP with those of severe PAP by integrating clinical findings, tissue pathology, and chest CT images. RESULTS: pCLE images of PAP showed giant cells with strong fluorescence, amorphous substances, and thin alveolar walls. Images of affected lesions in mild PAP were equivalent to those obtained in arbitrary lung lesions in severe cases. All 3 patients with mild PAP spontaneously improved or remained stable after ≥3 years of follow-up. Serum autoantibodies to granulocyte-macrophage colony-stimulating factor were detected in all 5 cases. CONCLUSION: Optical biopsy using pCLE can yield specific diagnostic findings, even in patients with mild PAP. pCLE images of affected areas in mild and severe PAP showed similar findings, indicating that the dysfunction level of pathogenic alveolar macrophages in affected areas is similar between both disease intensities.

The Application of Probe-Based Confocal Laser Endomicroscopy as a Diagnostic Tool in Choledochoscopic Gallbladder-Preserving Surgery.

BACKGROUND AND AIMS: Choledochoscopic gallbladder-preserving surgery (CGPS) has the advantage of treating benign gallbladder diseases on the premise of gallbladder preservation. However, it has no reliable preoperative diagnosis if the gallbladder is benign. Probe-based confocal laser endomicroscopy (pCLE) can obtain real-time and clear endoscopic images at the cell level in vivo. It is widely used in the diagnosis of digestive system diseases, but not in gallbladder diseases yet. We applied these two technologies in a complementary way into the diagnosis of gallbladder diseases and thereby lifted the reliability of CGPS. METHODS: We retrospectively analyzed the total 28 patients with the indication of CGPS with intraoperative pCLE scan referred to the Second Affiliated Hospital of Baotou Medical College between October 2019 and July 2020. The intraoperative pCLE results were compared with the postoperative pathology in various gallbladder diseases. RESULTS: We compared the intraoperative pCLE diagnosis with the postoperative pathological diagnosis and found a complete match without exception in both sensitivity and specificity. CONCLUSIONS: Based on our investigation, pCLE can provide the same accuracy as the traditional pathology in the diagnosis of gallbladder diseases with the additional advantages like noninvasive, real time, and instancy. This study serves to validate the correlation between CLE and histology. It holds a broad prospect in the application of pCLE as an intraoperative diagnosis in CGPS.

Probe-based confocal laser endomicroscopy for the diagnosis of pancreatic ductal structures.

BACKGROUND AND AIM: The image-based diagnosis of pancreatic diseases can be difficult and requires pathological evaluation. Probe-based confocal laser endomicroscopy (pCLE) enables real-time observation of the microscopic tissue pattern of lesion and may be a useful assistance for the diagnosis. This study aimed to evaluate the feasibility and utility of pCLE for the diagnosis of pancreatic diseases. METHODS: Thirty patients who underwent endoscopic retrograde cholangiopancreatography with pCLE for the evaluation of indeterminate pancreatic diseases from June 2015 to October 2018 were included in this study. The pCLE findings were interpreted according to the Miami Classification. RESULTS: Among a total of 30 patients, 12, 10, 4, and 4 patients received the definitive diagnoses of pancreatic ductal adenocarcinoma (PDAC), main duct intrapapillary mucinous neoplasm, autoimmune pancreatitis, and chronic pancreatitis, respectively. The diagnostic accuracy of pCLE for PDAC and pancreatitis (96.7% and 93.3%, respectively) was higher than that of cytology (76.7% and 63.3%, respectively) (P = 0.0227 and 0.0048, respectively). The sensitivity of pCLE for PDAC was significantly higher (91.7%) than that of cytology (41.7%) (P = 0.0094). Moreover, the specificity of pCLE for pancreatitis was significantly higher than that of cytology (90.9% vs 50%; P = 0.0029). However, the diagnostic accuracies of pCLE and cytology for main duct intrapapillary mucinous neoplasm did not differ significantly (96.7% and 86.7%, respectively). CONCLUSIONS: Probe-based confocal laser endomicroscopy may be effective for the diagnosis of pancreatic diseases as adjunct modality. It requires technical learning and further evaluation of its usefulness.

Probe-based confocal laser endomicroscopy for pleural malignancies diagnosis.

BACKGROUND AND OBJECTIVE: Probe based confocal laser endomicroscopy (pCLE) is an optical imaging technique allowing live tissue imaging at a cellular level. Currently, this tool remains experimental. Two studies regarding pleural disease have been published and suggest that pCLE could be valuable for pleural disease investigations. However, normal and malignant pleural pCLE features remain unknown. Therefore, we conducted a prospective trial of pCLE during medical thoracoscopy to study and describe the malignant and benign pleural pCLE features. METHODS: Every patient >18 years referred to our department for medical thoracoscopy was eligible. Medical thoracoscopy was performed under sedation, allowing spontaneous breathing. Five millilitres of fluorescein (10%) was intravenously administrated 5 min before image acquisition. The pCLE was introduced through the working channel of the thoracoscope and gently placed on the parietal pleura to record videos. Afterwards, biopsies were performed on the corresponding sites. Malignant and benign pleural pCLE features were precisely described and compared using 11 preselected criteria. RESULTS: A total of 62 patients were included in the analysis including 36 benign and 26 malignant pleura. Among our preselected criteria, 'abnormal tissue architecture' and 'dysplastic vessels' were strongly associated with malignancies (100% and 85% ss, 721% and 74% sp, respectively) whereas, the 'full chia seeds sign' and 'cell shape homogeneity' were associated with benignity (36% and 56% ss, 100% and 70% sp, respectively). No study-related adverse events occurred. CONCLUSION: Benign and malignant pleural involvement have clearly distinct pCLE features.

Cresyl violet as a new contrast agent in probe-based confocal laser endomicroscopy for in vivo diagnosis of gastric intestinal metaplasia.

BACKGROUND AND AIM: Cresyl violet (CV) is a topical dye that allows simultaneous chromoendoscopy and in vivo confocal laser endomicroscopy in identification of neoplastic changes of the lower gastrointestinal tract without intravenous injection of fluorescein, but as yet no investigation has reported its application in the diagnosis of gastric intestinal metaplasia (GIM). This study aims to assess the feasibility as well as diagnosis accuracy of topical CV for in vivo diagnosis of GIM by using probe-based confocal laser endomicroscopy (pCLE). METHODS: In this prospective, open-label, feasibility study, 129 confocal videos from 22 patients with known GIM were analyzed and compared with corresponding histological images to establish the CV staining characteristics. In addition, 47 patients with known or suspected GIM were prospectively enrolled to evaluate the accuracy of this topical CV endomicroscopic imaging. RESULTS: Probe-based confocal laser endomicroscopy with topical CV enabled clear visualization of the goblet cells, absorptive cells, and intestinal villi of GIM. The accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of pCLE diagnosis of GIM on a per-location analysis was 93.01%, 91.95%, 93.51%, 86.96%, and 96.11%, respectively. The intraclass correlation coefficient for inter-observer agreement and mean kappa value for intra-observer agreement for the diagnosis of GIM was 0.82 and 0.87, respectively. CONCLUSIONS: Topical CV enables real-time chromoendoscopy in conjunction with pCLE examination of the stomach and warrants accurate diagnosis of GIM. It may be an acceptable and potentially alternative dye for confocal imaging in the future.

Intraoperative confocal laser endomicroscopy for real-time in vivo tissue characterization during surgical procedures.

INTRODUCTION: Probe-based confocal laser endomicroscopy (pCLE) is an innovative technique providing real-time, in vivo optical biopsies. A previous ex vivo phase of the study (PERSEE) allowed identifying accurate pCLE criteria for the diagnosis of hepatic and peritoneal surgical specimens. This study aimed at evaluating the pCLE role for in vivo intra-abdominal tissue characterization during digestive cancer surgical procedures. METHODS: Between October 2014 and July 2015, consecutive patients diagnosed with digestive cancers and scheduled for a surgical resection or an exploratory laparoscopy were prospectively enrolled. Endomicroscopic images were acquired using a motorized Confocal Miniprobe™ with a bending distal tip providing easy access to abdominal organs. It was connected to an endomicroscopy system that allowed near-infrared illumination (at a wavelength of 785 nm) in conjunction with indocyanine green for contrast agent. A live audiovisual transmission was established between the surgeon and the pathologist for real-time interpretation of optical biopsies. Intraoperative pCLE performance for the diagnosis of suspicious nodules was assessed using corresponding surgical histopathology as reference standard. RESULTS: 21 consecutive patients were successfully enrolled. Live audiovisual transmission between the surgeon and the pathologist was successfully established in all cases. 62 pCLE sequences were acquired from different tissues [peritoneum (n = 27), liver (n = 21), lymph node (n = 4), diaphragm (n = 3), colon (n = 3), stomach (n = 2), and adrenal gland (n = 2)]. Malignant tissues were identified by fluorescently enhanced irregular cancerous tubes contrasting with dark glandular lumen and extracellular matrix. pCLE sensitivities and specificities were 67% and 100%, and 38% and 100% for peritoneal and hepatic carcinogenesis, respectively. One benign incident was reported during the trial with no patient consequence. CONCLUSIONS: Real-time intraoperative pCLE with near-infrared illumination is feasible and safe, provides additional information in terms of tissue characterization, and, in combination with telepathology, allows interactive collaboration between the surgeon and the pathologist during surgical procedures. Trial registration clinicaltrials.gov Identifier: NCT02312167.

In vivo probe-based confocal laser endomicroscopy in chronic interstitial lung diseases: Specific descriptors and correlation with chest CT.

BACKGROUND AND OBJECTIVE: Probe-based confocal laser endomicroscopy (pCLE) enables in vivo microimaging of the distal lung, during bronchoscopy. This study aims at identifying pCLE descriptors of chronic interstitial lung diseases (ILD), their correlations with chest HRCT and assessing inter-observer agreement. METHODS: pCLE was performed in 21 healthy volunteers (HV) and 59 non-smoking ILD patients, including 19 patients with idiopathic pulmonary fibrosis (IPF) or asbestosis, 15 with connective tissue disease-associated ILD (CTD-ILD), 17 with sarcoidosis and 8 with hypersensitivity pneumonitis (HP). pCLE descriptors were identified in ILD on the basis of comparison with HV. RESULTS: Nine pCLE descriptors were more frequent in ILD compared to HV, with good inter-observer agreement, including fluorescent bronchiolar cells (sarcoidosis, CTD-ILD and HP), fluorescent alveolar cells (CTD-ILD and HP), small alveolar entrance rings (IPF or asbestosis and CTD-ILD), enlarged axial elastic fibres (IPF or asbestosis), septal fibres (IPF or asbestosis, CTD-ILD and HP), disorganized acinar network and rigid acinar network (IPF or asbestosis and CTD-ILD), dense elastic network (IPF or asbestosis) and alveolar fluorescent nodular structures (in sarcoidosis) (P < 0.01, Fisher's exact test, all comparisons). The distribution of nodules on computed tomography (CT) appeared to correlate with pCLE alveolar nodular structures, rigid acinar network and septal fibres, while reticulations were associated with septal fibres and disorganized or dense acinar network; ground-glass opacities on CT with small alveolar entrances, rigid elastic network and septal fibres; and honeycombing with septal fibres. CONCLUSION: In the four groups of ILD studied, 9 pCLE descriptors are described, which appear specific and reproducible, and correlate with chest HRCT patterns.

Intraoperative Probe-Based Confocal Endomicroscopy to Histologically Differentiate Thyroid From Parathyroid Tissue Before Resection.

BACKGROUND: Frozen section is the standard method to histologically distinguish parathyroid tissue from thyroid tissue during endocrine neck surgery. Frozen section can be time-consuming and costly. Its drawback is that it is to be performed only after the removal of a suspected pathological tissue. This study demonstrates the use of probe-based confocal laser endomicroscopy (pCLE) to confirm histology prior to tissue resection. DESIGN: A prospective, single-institution, nonrandomized study was conducted. No sample size calculation was performed for this observational trial. The primary objective was the description of histological rendering of normal and pathological tissues through pCLE. Real-time in vivo fluorescence microscopy imaging was performed with the CystoFlex UHD probe after intravenous injection of 2.5 mL of 10% fluorescein sodium. RESULTS: Eleven patients with hyperparathyroidism and thyroid conditions were included. A total of 104 videos showing thyroid, parathyroid, adipose tissue, muscle, laryngeal nerve, and lymph nodes were recorded. Videos were compared with visual information and pathological samples (when sampling was indicated). Thyroid tissue could be identified based on the presence of colloid follicles (intensely fluorescent area surrounded by a small ridge of low-fluorescence epithelial cells) including the pathognomonic aspect of resorption vacuole. Parathyroid tissue could be identified based on a regular, "diamond-shaped" capillary network encompassing parathyroid chief cells. Blinded reinterpretation of pCLE videos demonstrated an 89.3% sensitivity and a 90% specificity as compared with histology in tissue recognition. CONCLUSION: This pilot study describes representative renderings of intraoperative pCLE to nontraumatically differentiate thyroid, parathyroid, and lymph nodes before surgical removal.

Individual probe based confocal laser endomicroscopy criteria in the analysis of indeterminate biliary strictures.

OBJECTIVES: Evaluation of indeterminate biliary strictures remains challenging due to limited sensitivity of endoscopic tissue sampling. Biliary probe-based confocal laser endomicroscopy (pCLE) has shown promise to detect and exclude neoplasia. However, knowledge of whether individual inflammatory criteria are more prevalent in neoplasia compared to benign strictures is limited. The objective of this work is to improve diagnosis of neoplastic and inflammatory conditions using pCLE. MATERIALS AND METHODS: The charts of all patients who underwent pCLE at a single referral center between 2009 and 2015 were reviewed. ERCP reports were reviewed for eleven Miami and Paris criteria. Primary outcome was the identification of neoplasia by histopathology (defined as high-grade dysplasia and/or adenocarcinoma). To model predictors of neoplasia, we fit a binary regression model incorporating data from pCLE operating criteria, pCLE impression, and PSC status. RESULTS: 97 patients were identified. In the 27 patients with neoplasia, there was increasing number of Miami malignant criteria (Pearson r = 0.512, p < .001) while inflammatory criteria were less prevalent. 10% (5/51, p < .001) of patients with benign pCLE impression developed neoplasia, while 48% (22/46, p < .001) with suspicious pCLE impressions developed neoplasia. The binary regression model to predict neoplasia had a sensitivity of 83.3%, specificity of 92.5%, and overall accuracy 89.7%. CONCLUSIONS: Presence of malignant criteria and absence of certain inflammatory criteria are more prevalent in patients with neoplasia. Our model, which weights individual imaging components, shows impressive sensitivity and specificity over prior prognostic efforts. Prospective studies will be required to evaluate this model.

Pilot study on probe-based confocal laser endomicroscopy for colorectal neoplasms: an initial experience in Japan.

BACKGROUND: The aim of this pilot study is to investigate the diagnostic yield of probe-based confocal laser endomicroscopy (pCLE) in the evaluation of depth of invasion in colorectal lesions. METHODS: Patients with colorectal lesions eligible for either endoscopic treatment or surgery were enrolled in the study. Tumor's depth of invasion was classified as mucosal or slight submucosal (M-SM1) and deep submucosal invasion or deeper (SM2 or deeper). White light endoscopy (WLE), magnifying narrow band imaging (M-NBI), and magnifying chromoendoscopy (M-CE) were used to assess colorectal lesions, and pCLE was used to identify tumor's features related to SM2 or deeper. The diagnostic classification of depth of invasion was obtained by correlating pCLE findings with histology results (on-site diagnosis). All colorectal lesions were stratified by a second endoscopist who was blinded to any clinical and histological information with the use of WLE, M-NBI, M-CE, and pCLE (off-line review). RESULTS: A total of 22 colorectal lesions were analyzed: seven were adenoma, ten intramucosal cancer, and five SM2 or deeper cancer. With respect to pCLE findings, loss of crypt structure was seen in all SM2 or deeper cancers and only in one M-SM1 lesion. Sensitivity, specificity, and accuracy of WLE, M-NBI, and M-CE in off-line review were 60/94/86, 60/94/86, and 80/94/91%, respectively. Sensitivity/specificity/accuracy of pCLE in off-line review were 80/94/91%, respectively. The inter-observer agreement of pCLE between on-site diagnosis and off-line review was 0.64 (95%CI 0.27-1.0). CONCLUSIONS: pCLE may represent a useful tool to evaluate the depth of invasion in colorectal lesions.

Feasibility of real-time intestinal bloodstream evaluation using probe-based confocal laser endomicroscopy in a porcine intestinal ischemia model.

BACKGROUND: Intestinal ischemia can lead to fatal complications if left unrecognized during surgery. The current techniques of intraoperative microvascular assessment remain subjective. Probe-based confocal laser endomicroscopy (pCLE) has the potential to objectively evaluate microvascular blood flow in real-time setting. The present study evaluated the technical feasibility of real-time intestinal bloodstream evaluation using pCLE in a porcine intestinal ischemia model. METHODS: Seven pigs were used. The intestinal ischemia model was prepared by sequentially dividing the mesenteric blood vessels. The intestinal bloodstream was evaluated on its serosal surface using pCLE (Cellvizio 488 probe, Ultra Mini O) at every 1-cm segment from a vessel-preservation border (i.e., the cut end of the vessel). Images of the blood vessels and flow of red blood cells (RBCs) in each visualized vessel were semi-qualitatively assessed using a 3-scale scoring system. In addition, 25 surgeons blindly assessed the 10 movies recorded at 0, 1, 2, 3, and 5 cm from a vessel-preservation border using a 4-scale scoring system to confirm the consistency of the evaluation of the pCLE system. RESULTS: Images of the blood vessels were successfully obtained from the cut end of the vessel to the segment 4 cm away. Good unidirectional flow of RBCs was observed from the cut end to the 2-cm segment, whereas the flow became bidirectional between 2 and 3 cm segments. Beyond 4 cm, no flow images were obtained. The specimen obtained from the segment beyond 4 cm showed remarkable mucosal color change, which was confirmed as a necrotic change histologically. The evaluations from the cut end of the vessel to the segment 1 cm away by surgeons were excellent or good and it was almost consistent. CONCLUSIONS: Real-time bloodstream evaluation using pCLE is feasible and potentially effective for predicting intestinal ischemia during surgery.

In vivo diagnosis of early-stage gastric cancer found after Helicobacter pylori eradication using probe-based confocal laser endomicroscopy.

BACKGROUND AND AIM: Early-stage gastric cancer (EGC) found after Helicobacter pylori (Hp) eradication often displays non-tumorous regenerative epithelium and/or maturated tumorous epithelium overlying the cancerous tissue, which may confuse endoscopic and histological diagnosis. Probe-based confocal laser endomicroscopy (pCLE) enables in vivo real-time optical biopsy. We compared the diagnostic yields for these EGC cases using conventional white light endoscopy (WL), magnifying endoscopy with narrow-band imaging (ME-NBI), pCLE, and endoscopic biopsy; we also compared the accuracy of the horizontal extent diagnosis between ME-NBI and pCLE. METHODS: This study enrolled 30 patients with 36 EGC lesions after successful Hp eradication. Diagnostic yields of WL, ME-NBI, pCLE, and endoscopic biopsy were prospectively compared. Four points of cancerous margins (oral, anal, anterior, and posterior sites) were also prospectively evaluated with M-NBI and pCLE to determine the horizontal extent of the EGC. RESULTS: Diagnostic yield was significantly higher with pCLE than with WL and endoscopic biopsy (97 vs 72%, 97 vs 72%, P = 0.0159, 0.0077, respectively), whereas it did not differ from ME-NBI (88.9%, P = 0.371). Height of non-tumorous regenerative epithelium or maturated atypical glands was 104.7 ± 34.2 µm in the pCLE-positive cases, whereas it was 188.3 ± 27.1 µm in a pCLE-negative case (P = 0.0004). Diagnostic accuracy of the horizontal margin of EGC was significantly higher with pCLE than with ME-NBI (92 vs 70%, P = 0.0159). CONCLUSION: pCLE may be helpful for the diagnosis of ambiguous ECG found after Hp eradication because it enables real-time scanning throughout the lesion and detection of subsurface microstructure.

Probe-based confocal laser endomicroscopy in the margin delineation of early gastric cancer for endoscopic submucosal dissection.

BACKGROUND AND AIM: We evaluated probe-based confocal laser endomicroscopy (pCLE) in the margin delineation of early gastric cancer (EGC) for endoscopic submucosal dissection in comparison with white-light imaging with chromoendoscopy (CE). METHODS: We conducted a prospective, randomized controlled study from November 2013 to October 2014 in a tertiary referral hospital. A total of 101 patients scheduled for endoscopic submucosal dissection due to differentiated EGC were randomized into pCLE and CE groups (pCLE 51, CE 50). Markings were made by electrocautery at the proximal and distal tumor margins, as determined by either pCLE or CE. The distance from the marking to the tumor margin was measured in the resected specimen histopathologically and was compared between the two groups by a linear mixed model. RESULTS: Among 104 lesions, 80 lesions with 149 markings (pCLE 68, CE 81) were analyzed after excluding undifferentiated EGCs (n = 8) and unidentifiable markings (n = 13). Although the complete resection rate showed no difference between the groups (94.6% vs 93.2%, P = 1.000), the median distance from the marking to the margin was shorter in the pCLE group (1.3 vs 1.8 mm, P = 0.525) and the proportion of the distance <1 mm was higher (43.9% vs 27.6%, P = 0.023) in the pCLE group. Finally, subgroup analysis with superficial flat lesions (18 lesions, 31 marking dots) showed a significantly decreased distance in the pCLE group (0.5 vs 3.1 mm, P = 0.007). CONCLUSIONS: Among EGCs with superficial flat morphology, in which the accurate evaluation of lateral extent is difficult with CE, pCLE would be useful for more precise margin delineation.

Probe-based confocal laser endomicroscopy for evaluating the submucosal invasion of colorectal neoplasms.

BACKGROUND: Probe-based confocal laser endomicroscopy (pCLE) is a novel method for in vivo histological analysis of colorectal neoplasm mucosa, which provides meaningful information for the development of adequate therapeutic strategies. However, the in vivo histology of colorectal neoplasm submucosa has not been studied. We assessed the feasibility and safety of pCLE for evaluating colorectal submucosa, and identified and validated diagnostic criteria for submucosal carcinoma infiltration. METHODS: From March to July 2014, 83 pCLE videos of 51 lesions in 31 patients who underwent scheduled colonoscopic procedures for the removal of colorectal neoplasms were acquired consecutively. During the procedures, pCLE videos of the lesions and biopsy samples for histopathological analysis were acquired. Final histopathological results were used as the gold standard. RESULTS: Based on the confocal pattern, we classified colorectal submucosa findings as negative (superficial submucosa, deep submucosa, and submucosa with fibrosis) or indicative of carcinoma infiltration. Dark and irregular cell nests with irregular cell architecture and little or no mucin were seen in submucosal carcinoma infiltration. Based on rates of correlation with pathological findings, the sensitivity, specificity, and accuracy of the classification of submucosal carcinoma infiltration by two observers were 91.7, 86.8, and 88.0 %, respectively. In addition, the results showed good interobserver agreement for the detection of submucosal carcinoma infiltration (κ = 0.757, standard error = 0.102). No adverse events occurred during the procedures. CONCLUSIONS: Submucosa assessment by pCLE is feasible and safe. pCLE is useful for the differentiation of normal submucosa from carcinoma infiltration, particularly when infiltration is accompanied by severe fibrosis. Large-scale prospective studies are needed to further evaluate the clinical impact of the use of pCLE during endoscopy.

Probe-based confocal laser endomicroscopy (pCLE): a preclinical investigation of the male genital tract.

The aim of this study was to assess the potential of probe-based confocal laser endomicroscopy (pCLE) as a new diagnostic imaging technique for the male genital tract. For this purpose, testes, epididymides, and vasa deferentia were obtained during transsexual surgery of healthy patients (n = 10, 26-52 years). Prior to this, testes of rats (n = 10, Sprague-Dawley) and mice (n = 8, wild-type) were examined. Ex vivo tissues were investigated by pCLE after topical fluorescence staining. Images and pCLE real-time video sequences were compared to images acquired by confocal laser scanning microscopy (CLSM); this allowed the identifying of corresponding microstructures. Interestingly, the seminiferous tubules of transsexual humans contained mainly spermatogonia due to long-term estrogen treatment, whereas the seminiferous tubules of the murine and rat spermatogenesis-related cell types were differentiated. Mosaicking improved the inspection potential by wide-angle views. Similarly, the microarchitecture of the epididymis and the vas deferens was successfully visualized in situ and on a cellular level by pCLE. In summary, pCLE allows for real-time identification of relevant microstructures responsible for spermatogenesis under ex vivo conditions. Additionally, pCLE enabled to localize vital spermatozoa in the testis thus opening up new ways to improve sperm retrieval rates during assisted reproduction. Both clinically relevant experiences hold promise to introduce this diagnostic method into a clinical study, and to investigate its potential as a clinical diagnostic procedure to expedite and improve the medical situation.

Development of a new classification for in vivo diagnosis of duodenal epithelial tumors with confocal laser endomicroscopy: A pilot study.

BACKGROUND AND AIM: Confocal laser endomicroscopy (CLE) has been established for in vivo diagnosis of various gastrointestinal diseases. However, validated criteria for confocal diagnosis of duodenal tumors do not exist. Therefore, the aim of the present pilot study was to develop a novel classification for in vivo optical diagnosis of duodenal tumors using CLE. METHODS: Consecutive patients with duodenal tumorous lesions were included. First, an initial classification system was developed which was then validated. Histopathology was used as a reference standard. RESULTS: A simple classification system for in vivo diagnosis of duodenal epithelial tumors using CLE was developed. Sensitivity, specificity, and accuracy were 90%, 100%, and 97%, respectively. Positive and negative predictive values were calculated as 100% and 96%. The kappa coefficient representing consistency was 1 between observers and within each observer. CONCLUSION: A new classification for in vivo diagnosis of duodenal epithelial tumors using confocal imaging has been developed. The new classification system allows adequate prediction of histology and could therefore be used to guide subsequent therapy of duodenal lesions.

Pre- and post-training session evaluation for interobserver agreement and diagnostic accuracy of probe-based confocal laser endomicroscopy for biliary strictures.

BACKGROUND AND AIM: Current diagnostic modalities for indeterminate biliary strictures offer low accuracy. Probe-based confocal laser endomicroscopy (pCLE) permits microscopic assessment of mucosal structures by obtaining real-time high-resolution images of the mucosal layers of the gastrointestinal tract. Previously, an interobserver study demonstrated poor to fair agreement even among experienced confocal endomicroscopy operators. Our objective was to assess interobserver agreement and diagnostic accuracy upon completion of a pCLE training session. METHODS: Forty de-identified pCLE video clips of indeterminate biliary strictures were sent to five endoscopists at four tertiary care centers for scoring. Observers subsequently attended a teaching session by an expert pCLE user that included 20 training clips and rescored the same pCLE video clips, which were randomized and renumbered. RESULTS: Pre-training interobserver agreement for all observers was 'fair' (Κ: 0.31, P-value: <0.0001) and diagnostic accuracy was 72% (55-80%). Post-training interobserver agreement for all observers was 'substantial' (Κ: 0.74, P-value: <0.0001) and diagnostic accuracy was 89% (80-95%). Using a paired t-test, we observed an increase of 17% (95% CI 7.6-26.4) in post-training diagnostic accuracy (t = 5.01, df = 4, P-value 0.007). CONCLUSIONS: Interobserver agreement and diagnostic accuracy improved after observers underwent training by an expert pCLE user with a specific sequence set. Users should participate in such training programs to maximize diagnostic accuracy of pCLE evaluation.

XRelevanceCAM: towards explainable tissue characterization with improved localisation of pathological structures in probe-based confocal laser endomicroscopy.

PURPOSE: Probe-based confocal laser endomicroscopy (pCLE) enables intraoperative tissue characterization with improved resection rates of brain tumours. Although a plethora of deep learning models have been developed for automating tissue characterization, their lack of transparency is a concern. To tackle this issue, techniques like Class Activation Map (CAM) and its variations highlight image regions related to model decisions. However, they often fall short of providing human-interpretable visual explanations for surgical decision support, primarily due to the shattered gradient problem or insufficient theoretical underpinning. METHODS: In this paper, we introduce XRelevanceCAM, an explanation method rooted in a better backpropagation approach, incorporating sensitivity and conservation axioms. This enhanced method offers greater theoretical foundation and effectively mitigates the shattered gradient issue when compared to other CAM variants. RESULTS: Qualitative and quantitative evaluations are based on ex vivo pCLE data of brain tumours. XRelevanceCAM effectively highlights clinically relevant areas that characterize the tissue type. Specifically, it yields a remarkable 56% improvement over our closest baseline, RelevanceCAM, in the network's shallowest layer as measured by the mean Intersection over Union (mIoU) metric based on ground-truth annotations (from 18 to 28.07%). Furthermore, a 6% improvement in mIoU is observed when generating the final saliency map from all network layers. CONCLUSION: We introduce a new CAM variation, XRelevanceCAM, for precise identification of clinically important structures in pCLE data. This can aid introperative decision support in brain tumour resection surgery, as validated in our performance study.

A novel contact optimization algorithm for endomicroscopic surface scanning.

PURPOSE: Probe-based confocal laser endomicroscopy (pCLE) offers real-time, cell-level imaging and holds promise for early cancer diagnosis. However, a large area surface scanning for image acquisition is needed to overcome the limitation of field-of-view. Obtaining high-quality images during scanning requires maintaining a stable contact distance between the tissue and probe. This work presents a novel contact optimization algorithm to acquire high-quality pCLE images. METHODS: The contact optimization algorithm, based on swarm intelligence of whale optimization algorithm, is designed to optimize the probe position, according to the quality of the image acquired by probe. An accurate image quality assessment of total co-occurrence entropy is introduced to evaluate the pCLE image quality. The algorithm aims to maintain a consistent probe-tissue contact, resulting in high-quality images acquisition. RESULTS: Scanning experiments on sponge, ex vivo swine skin tissue and stomach tissue demonstrate the effectiveness of the contact optimization algorithm. Scanning results of the sponge with three different trajectories (spiral trajectory, circle trajectory, and raster trajectory) reveal high-quality mosaics with clear details in every part of the image and no blurred sections. CONCLUSION: The contact optimization algorithm successfully identifies the optimal distance between probe and tissue, improving the quality of pCLE images. Experimental results confirm the high potential of this method in endomicroscopic surface scanning.

A novel Dual-Branch Asymmetric Encoder-Decoder Segmentation Network for accurate colonic crypt segmentation.

Colorectal cancer (CRC) is a leading cause of cancer-related deaths, with colonic crypts (CC) being crucial in its development. Accurate segmentation of CC is essential for decisions CRC and developing diagnostic strategies. However, colonic crypts' blurred boundaries and morphological diversity bring substantial challenges for automatic segmentation. To mitigate this problem, we proposed the Dual-Branch Asymmetric Encoder-Decoder Segmentation Network (DAUNet), a novel and efficient model tailored for confocal laser endomicroscopy (CLE) CC images. In DAUNet, we crafted a dual-branch feature extraction module (DFEM), employing Focus operations and dense depth-wise separable convolution (DDSC) to extract multiscale features, boosting semantic understanding and coping with the morphological diversity of CC. We also introduced the feature fusion guided module (FFGM) to adaptively combine features from both branches using cross-group spatial and channel attention to improve the model representation in focusing on specific lesion features. These modules are seamlessly integrated into the encoder for effective multiscale information extraction and fusion, and DDSC is further introduced in the decoder to provide rich representations for precise segmentation. Moreover, the local multi-layer perceptron (LMLP) module is designed to decouple and recalibrate features through a local linear transformation that filters out the noise and refines features to provide edge-enriched representation. Experimental evaluations on two datasets demonstrate that the proposed method achieves Intersection over Union (IoU) scores of 81.54% and 84.83%, respectively, which are on par with state-of-the-art methods, exhibiting its effectiveness for CC segmentation. The proposed method holds great potential in assisting physicians with precise lesion localization and region analysis, thereby improving the diagnostic accuracy of CRC.