1、1 Operetta 高内涵成像技 术 2 Operetta 简介 HCA for everylab High Content Imaging System Ideal for multi-user and multi-department environment Automated workflow for unbiased results Designed for flexibility and ease for all applications 3 什么是高内涵 细胞分析 高 内涵 细胞分析技术是 全自 动、高通量荧光显微成像结合 多参数定量分析 的技术，在单次细胞实验中可获得大量数据，
2、包括： 细胞中靶标分子的 空 间分布 ； 单个细胞或细胞群体 荧光强度、形态学以及细胞纹理学等多参数定量分析 ； 鉴定、分离并检测不同细胞群体的分类分析。 4 高内涵 显微成像与荧光显微镜的区别 高通量全自 动显微成像： 拍 摄模式：宽场、共聚焦及明场 全自 动高通量成像：激光自动对焦、软件控制的高精密度载物台，一个 视野、一个通道拍摄一块 96孔板只需要 2-3min 5 20x long WD objective, HeLa cells stained with Alexa Fluor488 labeled anti tubulin antibody and DRAQ5 6 Mouse e
3、mbryonic stem cell colonies after 3 days of culturing imaged on Operetta Early mouse embryonic stem cell colonies growing on a fibroblast feeder layer imaged on Operetta 7 Yeast 100x Objective 8 将 图像信息转化为定量的数字信息 HCS多参数分析 受 体 内 化 9 高内涵 细胞分析 (HCS)与流式 细胞仪的区别 HCS可 对细胞进行分区域分析 HCS形 态学分析参数丰富 HCS可研究 细胞的亚细胞显
4、微结构，如线粒体、内质网等 HCS有 图像进行直观分析，缩短决策的时间 HCS通量高， 样本制备通量及检测通量 ？ = 10 A cell can tell us what? Fluorescence Quantitation Morphology Caculation Texture Analysis 11 Cell Morphology: Area Width Length Roundness Width/Length ratio Intensity: Mean Intensity Intensity Variation (stddev, CV%) Radial Intensity Dis
5、tribution Texture: SER 8 features Gabor 2 features Haralick 4 features Quantification of Spot occurance Fibre occurance Fibre alignment Patch occurance Smooth/irregular distribution Pattern occurance Degree of granularity Available Parameters for Cellular Function Our unique set of morphometric, int
6、ensity-based and texture parameters provides a comprehensive solution for quantifying e. g. cytoskeletal rearrangement and spindle formation 12 我可以用高内涵成像系 统做什么 ？ 细胞形态学分析 信号通路研究 蛋白表达分析 细胞毒性分析 细胞增殖、凋亡及细胞周期分析 干 细胞克隆分析 神 经细胞神经元检测 13 研 发 领域： 肿瘤研究 (细胞周期、细胞增殖与凋亡、细胞迁移等分析） 中枢神 经及疼痛研究（神经元分析） 心血管及代 谢疾病研究（动脉硬化分
7、析） 炎症研究 （ GPCR、信号通路研究） 过敏及哮喘研究（信号通路研究） 病毒研究（病毒滴度 测定，抗病毒药物筛选等） 高内涵成像技 术已经应用到各类疾病研究中 applied to reduce attrition rates 14 全自 动平场矫正 ， 无需人 为干预 与人工手 动平场矫正不同， Operetta采用了全自 动平场矫正模式，无需人为干 预，并且同时保留了 raw data no FFC FFC 15 明 场数字相差成像 （ Digital Phase Contrast） HeLa cells in brightfield mode Digital Phase Image
8、 allows Segmentation Enhanced Live Cell Capabilities High Content Screening System 观 察活 细 胞 不需要使用任何 荧 光染料，即可 对 明 场图 像 进 行分析 毒性更低 16 不需要使用任何染料即可完成活 细胞示踪实验，完全没有细胞毒性； 内容丰富，可完成 对视野内细胞分裂、细胞凋亡、细胞运动轨迹分析等； Get a first impression of the specimen 高内涵无 标记细胞示踪技术 17 Important to consider all aspects of high cont
9、ent process 高内涵 = 装在壳子里的 显微镜 ？ 1. 全自 动高速细胞成像 2. 全自 动数据分析 3. 数据管理及二次分析 18 Cancer Biology 19 Induction Apoptosis by Small Molecules Control 3 M Staurosporine 30 M Staurosporine 20 Control 3 M Staurosporine 30 M Staurosporine Induction Apoptosis by Small Molecules Caspase-3 activationNuclear fragmentat
10、ion Nuclear size21 Apoptosis Assay Results Multiparametric results 22 Cell Cycle Assay - Overview Equilibration Incubation 24 h Cell seeding Compound addition Fixation and staining 18 h Readout 3 h Labeling Imaging 23 Cell Cycle Assay Operetta Images Mitotic index Anti phosphohistone H3 antibody, la
11、belled with Alexa Fluor 647 S-phase Anti EdU antibody, labelled with Alexa Fluor 488 DNA content HOECHST 33342, combined with CellMask Blue 24 Cell Cycle Assay Results I DNA Content Distribution S 2N 4N G2/M G1 Mean fluorescence intensity plotted against the integrated fluorescence intensity of the
12、nuclear dye. The mean intensity of the nucleus is fairly constant during S-phase and the nuclear size increases During M-phase and early G1-phase the nuclear size decreases, resulting in higher mean intensity values. Cells with 2N DNA (G1-phase) and 4N DNA (G2-/M-phase) can be clearly distinguished.
13、 25 Cell Cycle Assay Results II Dose-response curves for Thymidine (left) and Nocodazole (right) effects on cell cycle phases. S-phase cells (EdU positive) are shown in green, M-phase cells (pHH3 positive) in red, G1-cells in gray and G2-cells in orange (both based on DNA intensity) Thymidine induce
14、d arrest in G1-phase Nocodazole led to an increase of cells in M-phase 26 Seed Image Analysis - No Mask Required Oris Cell Migration assay Adherent cell migration / cell invasion assays without need for transmembrane inserts (Boyden chambers) 27 Oris Cell Migration Assay Assay Oris 96-well Collagen
15、I coated plates with Oris Cell Seeding Stoppers; two different cell lines: HT-1080 MDA-MB-231 Incubation for 6 h; remove stoppers; Migration for 18 h; fixation Staining with DAPI and TRITC-phalloidin Measurement Quantify open area remaning MDA-MB-231 pre-migration MDA-MB-231 post-migration 28 Reliab
16、le segmentation of brightfield images PhenoLOGIC machine learning on brightfield images Set training regions Automated image segmentation by PhenoLOGIC machine-learning technology Automated segmentation of brightfield images using the Find Texture Regions building block in Harmony High Content Analy
17、sis Software Calculation of open area and cell area using the Calculate Morphology Properties building block Read-outs: open area, cell area, confluency Detection of migrating cells over several time points 020406080100036912150h 2h 18h 21h 23h 25h 26h 41hConfluency %Region mmOpen Area Cell Area Con
18、fluency29 Operetta利用 DPC拍 摄的明场图像，开发了强大的无标记细胞示踪（ Label-free Cell Tracking） 分析模 块。 Operetta 软件 Harmony活细胞无标记示踪结果，通过时序动态观察，软件可自动生成细胞运动的 movie 30 After selection of a time window all other wells, fields, timepoints are no longer accessible until the time window is removed Images of the selected Time Window are being cached and a movie can be played Right-click allows to write the movie to disk (WMV format) Get a first impression of the specimen Workflow Cellular Cinema
