Confocal laser endomicroscopy for gastric neoplasm

Confocal laser endomicroscopy(CLE)is a novel endoscopic modality that provides real-time histological information via high-resolution magnified view of the mucosa.CLE has a higher sensitivity,specificity,and diagnostic accuracy in detecting atrophic gastritis as compared to chromoendoscopy and narrow-band imaging.It can even predict low-grade and high-grade intraepithelial neoplasia by analyzing gastric pit patterns.CLE may have some advantages over the standard biopsy protocol,such as higher diagnostic yield and fewer biopsy requirements.Its diagnostic accuracy in detecting superficial gastric cancer is higher than that of white-light endoscopy.Inherent limitations,such as a narrow field of vision,can be surpassed by technological advancements and integration with other detection methods.Artificial intelligence holds promise in automated analysis of histopathological images.Thus,CLE can be helpful in screening for early gastric cancer and may help reduce the risk of complications from repeated biopsies,such as mucosal damage,bleeding,and infection.

Diagnosis of gastric intraepithelial neoplasia by narrow-band imaging and confocal laser endomicroscopy

AIM:To evaluate the diagnosis of different differentiated gastric intraepithelial neoplasia (IN) by magnifica-tion endoscopy combined with narrow-band imaging (ME-NBI) and confocal laser endomicroscopy (CLE). METHODS:Eligible patients with suspected gastric IN lesions previously diagnosed by endoscopy in secondary hospitals and scheduled for further diagnosis and tratment were recruited for this study. Excluded from the study were patients who had liver cirrhosis, impaired renal function, acute gastrointestinal (GI) bleeding, coagulopathy, esophageal varices, jaundice, and GI post-surgery. Also excluded were those who were pregnant, breastfeeding, were younger than 18 years old, or were unable to provide informed consent. All patients had all mucus and bile cleared from their stom-achs. They then received upper GI endoscopy. When a mucosal lesion is found during observation with whitelight imaging, the lesion is visualized using maximal magnification, employing gradual movement of the tip of the endoscope to bring the image into focus. Saved images are analyzed. Confocal images were evaluated by two endoscopists (Huang J and Li MY), who were familiar with CLE, blinded to the related information about the lesions, and asked to classify each lesion as either a low grade dysplasia (LGD) or high grade dysplasia (HGD) according to given criteria. The results were compared with the final histopathologic diagnosis. ME-NBI images were evaluated by two endoscopists (Lu ZS and Ling-Hu EQ) who were familiar with NBI, blinded to the related information about the lesions and CLE images, and were asked to classify each lesion as a LGD or HGD according to the "microvascular pattern and surface pattern" classification system. The results were compared with the final histopathologic diagnosis. RESULTS: The study included 32 pathology-proven low grade gastric IN and 26 pathology-proven high grade gastric IN that were detected with any of the modalities. CLE and ME-NBI enabled clear visualization of the vascular microsurface patterns and microvascular structures of the gastric mucosa. The accuracy of the CLE and the ME-NBI diagnosis was 88% (95% CI:78%-98%) and 81% (95% CI: 69%-93%), respectively. The kappa coefficient of agreement between the histopathology and the in vivo CLE imaging was 0.755; between the histopathology and the in vivo CLE imaging was 0.615. McNemar's test (binomial distribution used) indicated that the agreement was significant (P < 0.05). When patients were diagnosed by MENBI with CLE, the overall accuracy of the diagnosis was 86.21% (95% CI:73%-96%), and the kappa coefficient of agreement was 0.713, according to McNemar's test (P < 0.05). CONCLUSION:Higher diagnostic accuracy, sensitivityand specificity of CLE over ME-NBI indicate the feasibility of these two techniques for the efficacious diagnostic classification of gastric IN.

In vivo subsurface morphological and functional cellular and subcellular imaging of the gastrointestinal tract with confocal mini-microscopy

AIM： To evaluate a newly developed hand-held confocal probe for in vivo microscopic imaging of the complete gastrointestinal tract in rodents. METHODS： A novel rigid confocal probe （diameter 7 mm） was designed with optical features similar to the flexible endomicroscopy system for use in humans using a 488 nm single line laser for fluorophore excitation, Light emission was detected at 505 to 750 nm. The field of view was 475 μm × 475 μm. Optical slice thickness was 7 μm with a lateral resolution of 0.7 μm. Subsurface serial images at different depths （surface to 250 μm） were generated in real time at 1024 × 1024 pixels （0.8 frames/s） by placing the probe onto the tissue in gentle, stable contact. Tissue specimens were sampled for histopathological correlation.RESULTS： The esophagus, stomach, small and large intestine and meso, liver, pancreas and gall bladder were visualised in vivo at high resolution in n = 48 mice. Real time microscopic imaging with the confocal minimicroscopy probe was easy to achieve. The different staining protocols （fluorescein, acriflavine, FITC-labelled dextran and L. esculentum lectin） each highlighted specific aspects of the tissue, and in vivo imaging correlated excellently with conventional histology. In vivo blood flow monitoring added a functional quality to morphologic imaging.CONCLUSION： Confocal microscopy is feasible in vivo allowing the visualisation of the complete GI tract at high resolution even of subsurface tissue structures. The new confocal probe design evaluated in this study is compatible with laparoscopy and significantly expands the field of possible applications to intra-abdominal organs. It allows immediate testing of new in vivo staining and application options and therefore permits rapid transfer from animal studies to clinical use in patients.

Real time detection of antibody-antigen interaction using a laser scanning confocal imaging-surface plasmon resonance system

A laser scanning confocal imaging-surface plasmon resonance （LSCI-SPR） instrument integrated with a wavelength-dependent surface plasmon resonance （SPR） sensor and a laser scanning confocal microscopy （LSCM） is built to detect the bonding process of human IgG and fluorescent-labeled affinity purified antibodies in real time. The shifts of resonant wavelength at different reaction time stages are obtained by SPR, corresponding well with the changes of the fluorescence intensity collected by using LSCM. The instrument shows the merits of the combination and complementation of the SPR and LSCM, with such advantages as quantificational analysis, high spatial resolution and real time monitor, which are of great importance for practical applications in biosensor and life science.

Simultaneous Measurement of Neural Activities of Acute Mouse Hippocampal Slices Using Multi-Electrode Array System and Laser Confocal Calcium Imaging

Recently, non-invasive, real-time and multi-point measurement of neural activities has become possible by using a multi-electrode array (MEA). Another method for multi-point measurement is the fluorescent imaging technique using voltage indicator dyes or calcium indicator dyes. Especially, calcium imaging using fluorescent calcium indicator dyes is often more useful, because they exhibit larger changes in the fluorescence intensity than voltage indicator dyes and their fluorescence changes can be detect easily. Additionally, calcium signals play key roles in the brain function, such as the long-term potentiation (LTP) in the hippocampus, and calcium imaging can be a powerful tool to elucidate the brain function. In this study, we constructed a measurement apparatus combining the MEA system and laser confocal calcium imaging and simultaneously measured electric signals and calcium signals in acute mouse hippocampal slices. The obtained results showed the availability of the present method.

Effect of Hypericin on Confocal Imaging of Ca^(2+) Signaling in Cultured Human Retinal Pigment Epithelium

Purpose: To investigate the mechanism of the Ca2 + signaling in cultured human retinal pigment epithelial(RPE) cells with the protein kinase C(PKC) specific inhibitor-hypericin stimulation.Methods: Cultured human RPE cells were analyzed using the fluorescence Ca2+ dye fluo-3 AM and laser scanning confocal microscope(LSCM) after stimulation with 100nM phorbol 12-myristate 13-acetate(PMA) and (or)5 concentrations of hypericin(1, 2, 3, 4 and 5 μM).Results: The normal fluorescence in RPE cells was strong and distributed throughout the cells. The nucleus appeared to be more fluorescent than the cytoplasm. After stimulation with PMA alone or 5 concentrations of hypericin, a rapid decrease in flurescence intensity was observed. There was no obvious difference in decreased curve among 5concentrations. However, after stimulation with a 24 hr preincubation of PMA and 5 concentrations of hypericin, a further decrease was not observed.Conclusion: Fluo-3 AM appears to be a good indicator of the change in Ca2+ occurring in RPE cells and hypericin is a strong inhibitor of Ca2 + influx channel. Hypericin has potential as a therapeutic drug for proliferative vitreoretinopathy(PVR), the inhibitory effect on PVR might be caused by blocking the PKC activity and inhibiting Ca2+ influxpathway.

3D Confocal Raman Imaging of Oil-Rich Emulsion from Enzyme-Assisted Aqueous Extraction of Extruded Soybean Powder

The understanding of the structure morphology of oil-rich emulsion from enzyme-assisted extraction processing(EAEP)was a critical step to break the oil-rich emulsion structure in order to recover oil.Albeit EAEP method has been applied as an alternative way to conventional solvent extraction method,the structure morphology of oil-rich emulsion was still unclear.The current study aimed to investigate the structure morphology of oil-rich emulsion from EAEP using 3 D confocal Raman imaging technique.With increasing the enzymatic hydrolysis duration from 1 to 3 h,the stability of oil-rich emulsion was decreased as visualized in the 3 D confocal Raman images that the protein and oil were mixed together.The subsequent Raman spectrum analysis further revealed that the decreased stability of oil-rich emulsion was due to the protein aggregations via SS bonds or protein-lipid interactions.The conformational transfer in protein indicated the formation of a compact structure.

SSL Depth: self-supervised learning enables 16× speedup in confocal microscopy-based 3D surface imaging [Invited]

In scientific and industrial research, three-dimensional (3D) imaging, or depth measurement, is a critical tool that provides detailed insight into surface properties. Confocal microscopy, known for its precision in surface measurements, plays a key role in this field. However, 3D imaging based on confocal microscopy is often challenged by significant data requirements and slow measurement speeds. In this paper, we present a novel self-supervised learning algorithm called SSL Depth that overcomes these challenges. Specifically, our method exploits the feature learning capabilities of neural networks while avoiding the need for labeled data sets typically associated with supervised learning approaches. Through practical demonstrations on a commercially available confocal microscope, we find that our method not only maintains higher quality, but also significantly reduces the frequency of the z-axis sampling required for 3D imaging. This reduction results in a remarkable 16×measurement speed, with the potential for further acceleration in the future. Our methodological advance enables highly efficient and accurate 3D surface reconstructions, thereby expanding the potential applications of confocal microscopy in various scientific and industrial fields.

Development of a portable reflectance confocal microscope and its application in the noninvasive in vivo evaluation of mesenchymal stem cell-promoted cutaneous wound healing

The process of wound healing is routinely evaluated by histological evaluation in the clinic,which may cause scarring and secondary injury.Reflectance confocal microscopy(RCM)represents a noninvasive,real-time imaging technique that allows in vivo evaluation of the skin.Traditional RCM was wide-probe-based,which limited its application on uneven and covered skin.In this study,we report the development of a portable reflectance confocal microscope(PRCM)in which all components were assembled in a handheld shell.Although the size and weight of the PRCM were reduced based on the use of a microelectromechanical system,the resolution was kept at 0.91μm,and the field of view of the system was 343μm×532μm.When used in vivo,the PRCM was able to visualize cellular and nuclear morphology for both mouse and human skin.PRCM evaluations were then performed on wounds after topically applied mesenchymal stem cells(MSCs)or saline treatment.The PRCM allowed visualization of the formation of collagen bundles,re-epithelization from the wound edge to the wound bed,and hair follicle regeneration,which were consistent with histological evaluations.Therefore,we offer new insights into monitoring the effects of topically applied MSCs on the process of wound healing by using PRCM.This study illustrates that the newly developed PRCM represents a promising device for real-time,noninvasive monitoring of the dynamic process of wound healing,which demonstrates its potential to diagnose,monitor,or predict disease in clinical wound therapy.

Confocal Image 3D Surface Measurement with Optical Fiber Plate

A whole-field 3D surface measurement system for semiconductor wafer inspection is described.The system consists of an optical fiber plate,which can split the light beam into N^2 subbeams to realize the whole-field inspection.A special prism is used to separate the illumination light and signal light.This setup is characterized by high precision,high speed and simple structure.

Advanced gastrointestinal endoscopic imaging forinflammatory bowel diseases

Gastrointestinal luminal endoscopy is of paramount importance for diagnosis, monitoring and dysplasia surveillance in patients with both, Crohn's disease and ulcerative colitis. Moreover, with the recent recognition that mucosal healing is directly linked to the clinical outcome of patients with inflammatory bowel disorders, a growing demand exists for the precise, timely and detailed endoscopic assessment of superficial mucosal layer. Further, the novel field of molecular imaging has tremendously expanded the clinical utility and applications of modern endoscopy, now encompassing not only diagnosis, surveillance, and treatment but also the prediction of individual therapeutic responses. Within this review, we describe how novel endoscopic approaches and advanced endoscopic imaging methods such as high definition and high magnification endoscopy, dye-based and dye-less chromoendoscopy, confocal laser endomicroscopy, endocytoscopy and molecular imaging now allow for the precise and ultrastructural assessment of mucosal inflammation and describe the potential of these techniques for dysplasia detection.

Confocal laser endomicroscopy in the “in vivo” histological diagnosis of the gastrointestinal tract

Recent technological advances in miniaturization have allowed for a confocal scanning microscope to be integrated into a conventional flexible endoscope,or into trans-endoscopic probes,a technique now known as confocal endomicroscopy or confocal laser endomicroscopy.This newly-developed technology has enabled endoscopists to collect real-time in vivo histological images or "virtual biopsies" of the gastrointestinal mucosa during endoscopy,and has stimulated significant interest in the application of this technique in clinical gastroenterology.This review aims to evaluate the current data on the technical aspects and the utility of this new technology in clinical gastroenterology and its potential impact in the future,particularly in the screening or surveillance of gastrointestinal neoplasia.

High technology imaging in digestive endoscopy

A thorough endoscopic visualization of the digestive mucosa is essential for reaching an accurate diagnosis and to treat the different lesions. Standard white light endoscopes permit a good mucosa examination but, nowadays, the introduction of powerful endoscopic instrumentations increased ability to analyze the fi nest details. By applying dyes and zoom-magnifi cation endoscopy further architectural detail of the mucosa can be elucidated. New computed virtual chromoendoscopy have further enhanced optical capabilities for the evaluation of submucosal vascolar pattern. Recently, confocal endomicroscopy and endocytoscopy were proposed for the study of ultrastructural mucosa details. Because of the technological contents of powerful instrumentation, a good knowledge of implemented technologies is mandatory for the endoscopist, nowadays. Nevertheless, there is a big confusion about this topic. We will try to explain these technologies and to clarify this terminology.

Software for automated classification of probe-based confocal laser endomicroscopy videos of colorectal polyps

AIM:To support probe-based confocal laser endomi-croscopy (pCLE) diagnosis by designing software for the automated classification of colonic polyps. METHODS:Intravenous fluorescein pCLE imaging of colorectal lesions was performed on patients under-going screening and surveillance colonoscopies, followed by polypectomies. All resected specimens were reviewed by a reference gastrointestinal pathologist blinded to pCLE information. Histopathology was used as the criterion standard for the differentiation between neoplastic and non-neoplastic lesions. The pCLE video sequences, recorded for each polyp, were analyzed off-line by 2 expert endoscopists who were blinded to the endoscopic characteristics and histopathology. These pCLE videos, along with their histopathology diagnosis, were used to train the automated classification software which is a content-based image retrieval technique followed by k-nearest neighbor classification. The performance of the off-line diagnosis of pCLE videos established by the 2 expert endoscopists was compared with that of automated pCLE software classification. All evaluations were performed using leave-one-patient- out cross-validation to avoid bias. RESULTS:Colorectal lesions (135) were imaged in 71 patients. Based on histopathology, 93 of these 135 lesions were neoplastic and 42 were non-neoplastic. The study found no statistical significance for the difference between the performance of automated pCLE software classification (accuracy 89.6%, sensitivity 92.5%, specificity 83.3%, using leave-one-patient-out cross-validation) and the performance of the off-line diagnosis of pCLE videos established by the 2 expert endoscopists (accuracy 89.6%, sensitivity 91.4%, specificity 85.7%). There was very low power (< 6%) to detect the observed differences. The 95% confidence intervals for equivalence testing were:-0.073 to 0.073 for accuracy, -0.068 to 0.089 for sensitivity and -0.18 to 0.13 for specificity. The classification software proposed in this study is not a "black box" but an informative tool based on the query by example model that produces, as intermediate results, visually similar annotated videos that are directly interpretable by the endoscopist. CONCLUSION:The proposed software for automated classification of pCLE videos of colonic polyps achieves high performance, comparable to that of off-line diagnosis of pCLE videos established by expert endoscopists.

Molecular confocal laser endomicroscopy:A novel technique for in vivo cellular characterization of gastrointestinal lesions

While flexible endoscopy is essential for macroscopic evaluation,confocal laser endomicroscopy(CLE)has recently emerged as an endoscopic method enabling visualization at a cellular level.Two systems are currently available,one based on miniprobes that can be inserted via a conventional endoscope or via a needle guided by endoscopic ultrasound.The second system has a confocal microscope integrated into the distal part of an endoscope.By adding molecular probes like fluorescein conjugated antibodies or fluorescent peptides to this procedure(either topically or systemically administered during on-going endoscopy),a novel world of molecular evaluation opens up.The method of molecular CLE could potentially be used for estimating the expression of important receptors in carcinomas,subsequently resulting in immediate individualization of treatment regimens,but also for improving the diagnostic accuracy of endoscopic procedures by identifying otherwise invisible mucosal lesions.Furthermore,studies have shown that fluorescein labelled drugs can be used to estimate the affinity of the drug to a target organ,which probably can be correlated to the efficacy of the drug.However,several of the studies in this research field have been conducted in animal facilities or in vitro,while only a limited number of trials have actually been carried out in vivo.Therefore,safety issues still needs further evaluations.This review will present an overview of the implications and pitfalls,as well as future challenges of molecular CLE in gastrointestinal diseases.

Diagnostic imaging advances in murine models of colitis

Inflammatory bowel diseases(IBD) such as Crohn's disease and ulcerative colitis are chronic-remittent inflammatory disorders of the gastrointestinal tract still evoking challenging clinical diagnostic and therapeutic situations. Murine models of experimental colitis are a vital component of research into human IBD concerning questions of its complex pathogenesis or the evaluation of potential new drugs. To monitor the course of colitis, to the present day, classical parameters like histological tissue alterations or analysis of mucosal cytokine/chemokine expression often require euthanasia of animals. Recent advances mean revolutionary noninvasive imaging techniques for in vivo murine colitis diagnostics are increasingly available. These novel and emerging imaging techniques not only allow direct visualization of intestinal inflammation, but also enable molecular imaging and targeting of specific alterations of the inflamed murine mucosa. For the first time, in vivo imaging techniques allow for longitudinal examinations and evaluation of intra-individual therapeutic response. This review discusses the latest developments in the different fields of ultrasound, molecularly targeted contrast agent ultrasound, fluorescence endoscopy, confocal laser endomicroscopy as well as tomographic imaging with magnetic resonance imaging, computed tomography and fluorescence-mediated tomography,discussing their individual limitations and potential future diagnostic applications in the management of human patients with IBD.

Endoscopic imaging of Barrett's esophagus

The incidence of esophageal adenocarcinoma (EAC) has dramatically increased in the United States as well as Western European countries. The majority of esophageal adenocarcinomas arise from a backdrop of Barrett&rsquo;s esophagus (BE), a premalignant lesion that can lead to dysplasia and cancer. Because of the increased risk of EAC, GI society guidelines recommend endoscopic surveillance of patients with BE. The emphasis on early detection of dysplasia in BE through surveillance endoscopy has led to the development of advanced endoscopic imaging technologies. These techniques have the potential to both improve mucosal visualization and characterization and to detect small mucosal abnormalities which are difficult to identify with standard endoscopy. This review summarizes the advanced imaging technologies used in evaluation of BE.

Light and sound- emerging imaging techniques for inflammatory bowel disease

Patients with inflammatory bowel disease are known to have a high demand of recurrent evaluation for therapy and disease activity. Further, the risk of developing cancer during the disease progression is increasing from year to year. New, mostly non-radiant, quick to perform and quantitative methods are challenging, conventional endoscopy with biopsy as gold standard. Especially, new physical imaging approaches utilizing light and sound waves have facilitated the development of advanced functional and molecular modalities. Besides these advantages they hold the promise to predict personalized therapeutic responses and to spare frequent invasive procedures. Within this article we highlight their potential for initial diagnosis, assessment of disease activity and surveillance of cancer development in established techniques and recent advances such as wide-view full-spectrum endoscopy, chromoendoscopy, autofluorescence endoscopy, endocytoscopy, confocal laser endoscopy, multiphoton endoscopy, molecular imaging endoscopy, B-mode and Doppler ultrasound, contrast-enhanced ultrasound, ultrasound molecular imaging, and elastography.

Recent advances in targeted endoscopic imaging:Early detection of gastrointestinal neoplasms

Molecular imaging has emerged as a new discipline in gastrointestinal endoscopy.This technology encompasses modalities that can visualize disease-specific morphological or functional tissue changes based on the molecular signature of individual cells.Molecular imaging has several advantages including minimal damage to tissues,repetitive visualization,and utility for conducting quantitative analyses.Advancements in basic science coupled with endoscopy have made early detection of gastrointestinal cancer possible.Molecular imaging during gastrointestinal endoscopy requires thedevelopment of safe biomarkers and exogenous probes to detect molecular changes in cells with high specificity anda high signal-to-background ratio.Additionally,a high-resolution endoscope with an accurate wide-field viewing capability must be developed.Targeted endoscopic imaging is expected to improve early diagnosis and individual therapy of gastrointestinal cancer.

Dark-field line confocal imaging with point confocality and extended line field for bulk defects detection

Fabrication of high-quality optics puts a strong demand on high-throughput detection of macroscopic bulk defects in optical components.A dark-field line confocal imaging method is proposed with two distinct advantages:(ⅰ)a point-to-line confocal scheme formed by a columnar elliptical mirror and an optical fiber bundle breaks through the constraint on light collection angle and field of view in the traditional line confocal microscopy using an objective,allowing for an extended confocal line field of more than 100 mm while maintaining a light collection angle of 27°;(ⅱ)the bulk defects are independently illuminated as a function of time to eliminate the cross talk in the direction of the confocal slit,thus preserving point confocality and showing the optical section thicknesses to be 162μm in the axial direction,and 19 and 22μm in the orthogonal transverse directions.The experimental results verify that the method has a minimum detectable bulk defect of less than 5μm and an imaging efficiency of 400 mm2/s.The method shows great potential in high-throughput and highsensitivity bulk defects detection.