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.

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.

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.

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.

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.

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.

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.

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.

Simultaneous multi-parameter observation of Harring-tonine-treating HL-60 cells with both two-photon and confocal laser scanning microscopy

Harringtonine (HT), a kind of anticancer drug isolated from Chinese herb-Cephalotaxus hainanensis Li, can induce apoptosis in promyelocytic leukemia HL-60 cells. With both two-photon laser scanning microscopy and confocal laser scanning microscopy in combination with the fluores-cent probe Hoechst 33342, tetramethyrhodamine ethyl ester (TMRE) and Fluo 3-AM, we simulta-neously observed HT-induced changes in nuclear morphology, mitochondrial membrane potential and intracellular calcium concentration ([Ca2+]i) in HL-60 cells, and developed a real-time, sensitive and invasive method for simultaneous multi-parameter observation of drug- treating living cells at the level of single cell.

Femtosecond fiber laser at 780 nm for two-photon autofluorescence imaging

We present an Er-doped fiber(Er:fiber)-based femtosecond laser at 780 nm with 256 MHz repetition rate, 191 fs pulse duration, and over 1 W average power.Apart from the careful third-order dispersion management, we introduce moderate self-phase modulation to broaden the output spectrum of the Er:fiber amplifier and achieve 193 fs pulse duration and 2.43W average power.Over 40% frequency doubling efficiency is obtained by a periodically poled lithium niobate crystal.Delivering through a hollow-core photonic bandgap fiber, this robust laser becomes an ideal and convenient light source for two-photon autofluorescence imaging.

共聚焦显微成像系统P9000图像质量评价临床研究

目的评价共聚焦显微成像系统P9000检测人体消化道相关组织的有效性和安全性。方法选取2021年8月至2022年6月在北京中医药大学东方医院和山东大学齐鲁医院行内镜检查的96例患者为研究对象。94例受试者纳入全分析集(Full Analysis Set,FAS),90例患者纳入符合方案集(Per Protocol Set,PPS)。通过多中心、随机、自身对照研究,统计共聚焦显微成像系统P9000与国际公认的Cellvizio®系统检测人体消化道相关组织的图像质量评分一致性和95%CI,以及两组的操作时间和不良反应发生情况。结果FAS和PPS两款器械图像质量评分一致性和95%CI分别为92.55%(87.25%~97.86%)和92.22%(86.69%~97.76%),两者95%CI下限均大于目标值84.6%;两组器械操作时间相当,差异无统计学意义(P>0.05);两组均无设备导致的不良反应发生。结论共聚焦显微成像系统P9000的临床使用有效性和安全性达到了设计要求,值得临床推广应用。

基于共聚焦激光显微内镜的海水淹溺肺损伤大鼠肺通透性的实时活体评价

背景急性肺损伤是临床常见急危重症,其核心病理特征是气血屏障的破坏,目前对肺损伤中气血屏障结构及功能改变进行活体评估的研究甚少。目的使用共聚焦激光显微内镜实现大鼠肺的在体可视化成像和海水淹溺肺损伤大鼠肺通透性的实时动态活体评估。方法10只SD大鼠随机分为对照组和海水淹溺(seawater drowning,SW)组,每组5只;SW组通过气管滴注人工海水建立海水淹溺肺损伤模型,造模后3 h进行肺活体观察窗开窗手术,4 h时经股静脉推注0.2 mL异硫氰酸荧光素-葡聚糖(10 mg/mL);对照组不进行海水滴注,其余操作同SW组。使用共聚焦激光显微内镜对右肺下叶进行30 min活体观察,对成像0 min、10 min、20 min、30 min时的共聚焦图像进行染料渗漏指数及荧光分布均匀程度的分析;成像结束后处死大鼠,取肺组织、外周血及肺泡灌洗液,进行组织病理学检查、FITC-Dextran渗漏量检测、肺泡灌洗液蛋白含量检测及肺组织湿/干比测定。结果经静脉给予FITC-Dextran后,共聚焦激光显微内镜下可观察到肺微循环和肺泡结构的实时图像;对照组血管及肺泡结构较清晰,未出现血管外空间荧光强度的明显增加,而SW组血管结构随时间推移逐渐模糊,各时间点SW组的渗漏指数均高于对照组(P<0.05),10 min、20 min、30 min时荧光分布的变异系数均低于对照组(P<0.05);SW组的染料渗漏量、肺泡灌洗液蛋白含量及肺组织湿/干比均高于对照组(P<0.05)。结论海水淹溺造成大鼠肺组织结构破坏,肺通透性增加;利用共聚焦激光显微内镜可实现大鼠肺组织结构的在体可视化成像,通过对共聚焦图像的定量分析,还可实现大鼠气血屏障通透性的实时活体评估。

超广角长出瞳距共聚焦眼底成像系统光学设计(特邀)

眼底检查能有效筛查糖尿病、高血压等全身性疾病引发的眼底病变,是一种常规性检查。然而,当前的角膜接触式超广角高分辨率视网膜成像设备通常难以兼顾长工作距和免散瞳的功能,给患者造成了不适,也增加了检查难度。针对眼科检查中视网膜广域成像的需求,基于共聚焦激光扫描眼底成像技术,设计了一种超广角长出瞳距免散瞳的眼底成像光学系统。系统采用透射式结构,综合考虑长出瞳距和免散瞳的要求,实现了110°超广角成像。系统具备12 mm出瞳距,瞳孔处光束尺寸小于2 mm,并内置视度补偿镜组,可对−15~+15 D的屈光度人眼进行有效补偿。设计结果表明:系统的眼底物方分辨率为7.5μm,具备优良的成像质量,成像范围与工作距离满足了实际使用需求。该研究为超广角共聚焦眼底成像光学系统的设计提供了有益参考,对于提高眼底成像质量和临床诊断准确性具有一定意义。

激光扫描共聚焦光谱成像系统

在激光扫描共聚焦显微成像技术基础上引入了光谱成像技术以便区分生物组织中的不同荧光成分.采用分光棱镜对荧光进行光谱展开,在光谱谱面处设置两个可移动缝片形成出射狭缝,两个步进电机带动安装其上的两个缝片设置系统在整个工作波长（400～ 700 nm）内的光谱带宽,其最小光谱带宽优于5 nm.用488 nm激光和低压汞灯实际测量了几条谱线对应的狭缝位置并和理论值做了比较,结果显示实际狭缝位置和理论值的差值均小于0.1 mm.在全光谱和50 μm出射狭缝（对应2.5 nm光谱带宽）对老鼠肾脏组织进行了共聚焦光谱成像实验,获得了老鼠肾脏组织中DAPI标定的细胞核图像和Alexa Fluor（R） 488标定的肾脏小球曲管图像,实现了对老鼠肾脏组织不同成分的区分.实验结果表明：提出的系统能够进行共聚焦光谱成像,扩大了共聚焦显微镜的适用范围.

激光扫描共聚焦显微镜的基本原理及其使用技巧

介绍了激光扫描共聚焦显微镜(laser scanning confocal microscopy,LSCM)的原理和构造,分析了LSCM的优点和缺点,并以花粉为例,详细介绍了共聚焦针孔直径、光电倍增管检测器增益、激光强度、扫描速度、扫描方式和Z轴步距等重要参数设置对共聚焦成像的不同影响。探讨了正确使用LSCM的方法与技巧,如获取高质量的图像、图像保存及图像处理,以便为科技人员利用LSCM开展更多植物学与环境科学相关的研究提供参考。

激光共聚焦三维图像技术在油田中的应用

将激光共聚焦扫描显微镜(Laser Confocal Scanning Microscope)分析技术应用到大庆探区的储层研究中,进行了储层孔隙和微裂缝的三维结构重建,对生油岩中的有机质分布和储层中原油形态进行了详细研究,其中原油形态观察对薄差水淹层的研究有重要意义,研究了裂缝中次生包裹体特征及形成期次,此外,该方法应用于微体古生物化石研究在大庆探区发现了以前本区未见的孢子化石种类,在原油乳化机理研究方面,首次应用共聚焦观察了乳化油的内部结构,通过动态分析可以确定其稳定性,这为乳化油乳化机理研究提供了科学可靠的依据。

多孔介质微观孔隙结构三维成像技术

详细介绍目前国际上最先进的多孔介质微观结构三维成像技术,包括系列切片技术、聚焦离子束技术(FIB)、激光共聚焦扫描显微镜、Micro-CT技术,分析了各自的优越性及局限性,探讨了Micro-CT技术的应用现状及其在地质学领域的应用前景。分析结果表明,Micro-CT及今后的Nano-CT技术是一种无损伤的3D成像技术,不但可获得足够分辨率的多孔介质微观孔隙结构3D图像,而且还可以现场实时检测孔隙中流体的渗流状态,在多孔介质孔隙结构及渗流机理研究中具有重要的应用价值。该技术必定成为今后的主流技术,不仅将在石油工程、水文工程等领域发挥重要的作用,而且还将广泛应用于岩石学、矿物学、古生物学、材料学、资源科学及环境科学等领域。该技术的研究和应用在我国还处在起步阶段,应加大设备引进和研究力度。

ZEISS LSM 780激光扫描共聚焦显微镜的三维成像及分析

针对ZEISS LSM 780激光扫描共聚焦显微镜的三维图像获取步骤做了系统的介绍,详述了光学切片的设置和获取过程,后期应用Zen2011 black和blue软件进行三维重构的渲染及图像分析、数据采集。列举出三维成像中常见问题及解决办法,使仪器为教学和科研工作提供更优质的服务。

应用小动物活体激光共聚焦成像系统对大鼠腹壁循经组织组织液分布的初步观察

目的:初步观察探究活体大鼠腹壁循经组织的组织液分布特征和相关形态结构。方法:大鼠麻醉后尾静脉注射10%的荧光素钠溶液(0.1 ml/kg),注射30 min后剥离腹部皮肤,使用活体激光共聚焦成像系统观察腹壁组织不同深度的荧光素钠溶液分布及相关组织的形态结构特征。结果:荧光素钠注射30 min后,使用活体激光共聚焦成像系统观察到在大鼠腹壁正中线即任脉循行位置不同深度组织中的组织间隙较大,其中填充着大量混合荧光素钠的组织液,并分布有少量胶原纤维、毛细血管、脂肪细胞等实体组织。腹中线水平旁开5 mm和1 cm处的非经组织主要为实体组织,其组织中的间隙空间和组织液含量远远小于腹中线。新鲜腹壁组织冰冻切片显示,腹壁组织中存在大小不等、纵横交错且相互连接沟通的组织间隙,腹中线组织的间隙空间大于周围组织,这一结果与激光共聚焦图像特征吻合。结论:大鼠腹壁循任脉组织中存在比周围非经组织较大的组织间隙和较多的组织液。