应用培训_Operetta高内涵筛选系统汇总

1、perkinelmerfor the betterhuman health | environmental healthoperetta高内涵筛选系统应用培训黄晔2014.3ru2os （人骨肉瘤）cells expressing tagged endothelin （内皮素）a receptor, dose-response curves for endothelin 1 and sarafatoxin 6b u20s cells expressing tagged endothelin （内皮素）a receptor, dose-response curves for endothelin

2、 1 and sarafatoxin 6buo-leoo-su&l ou-moqs s=。o qenudc|perklnelmerarw the better什么是高内涵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，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。80e”ooe-ro6*34.0e+32.0ei3o.oe-o loe+6 2.0e+63.0 注 64.0&6integrated dnalntensityfperkln

3、elmer0rw the better高内涵能做什么?7fperklnelmer0rw the better预设分析方案，方便应用harmony即用解决方案应用举例细胞计数和细胞核计数细胞增殖、细胞毒性活细唧死细胞讦数细胞活力：洁细胸死细胞计数星化细葩核中的标记物细胞核中的蛋白质表达（例如.胱天蛋白酶3细胞周期一 5期昆化 du、edu）量化细胞质中的标记物细胞溶质中的蛋白质表达线粒体质量线粒体膜电位的损失磷脂质变异肝脏脂肪变性细胞周期:g2向m期的转变（细胞周期蛋白b1）量化细胞膜中的标记物质膜中的蛋白质表达信号通路：细胞膜皱缩细胞溶质向细胞核的转位信号通路：转录因子活化细胞溶质向细胞膜的转

4、位信号通路，细胞质向细胞膜的转位荧光重分布分析-细胞骨架细胞骨架重组分析蛋白点分析过氧化物薛体量化囊泡形成 gpcr活性：抑制再白补偿（如transfluoi）细胞核荧光分析荧光原位杂交（fish）核分析-核收缩核能大和收缩核断裂细胞凋亡/核断裂核分类-dna含量细胞周期：dna含量细胞形状-细胞变形细胞伸展, 细胞分离, 细胞国度变化有丝分裂指数细胞周期：有丝分裂指数（磷酸化组雷白h卦细胞周期分类磷酸化组蛋白阳、edu, dna含量和核形态受体内化gpcr活化；内香作用、标记配体内化和对ph敏感的染料的介导内化神经突分析神经突增生细胞克隆形成施时间形成的细胞克隆群落微核分析双核细胞中的微核量

5、化迁移迁移检测和划痕检测脂滴分析磷脂质异变、核内体和溶酶体分析细胞器分析基于纹理的分割干细胞群落,干细胞群落分析带和不带饲养层）.克隆群落形成显型分析细胞骨架：细胞骨架重组、细胞伸展/变圆、细胞毒性细胞凋亡nuclear fragmentationcaspase-3 activationcell seedingequilibrationincubationreadoutcompound additionfixation & staining16 hfperklnelmer0rw the better凋亡检测-成像caspase 3 活化细胞计数 细胞核大小 细胞核碎片化alexa fluor

6、488 labeled anti caspase-3 antibodydraq5draq5draq5control 20x long wd物镜成像增加星抱菌素浓导致caspase-3浓度增 加药物处理后的细胞核表现出典型的凋 亡现象，核皱缩，面积变小，亮度增 力口；在凋亡晚期细胞核碎片化3 pm staurosporine30 pm staurosporine12凋亡检测一结果perklnelmerarw the betterllog staurosporine pmec50 2.824【】 s-a)。9sod s_soldod细胞周期perklnelmer0rw the better14g1

7、 early s lates g2 mitosisincorporation of edupfh3f细胞周期检测成像perklnelmer0rw the better16false color overlay of hoechst 33342,alexa fluor 488 and alexa fluor 647 channel30 pm thymidinecell cycle phases for individual cellsmitotic indexanti phosphohistone h3 antibody, labeled with alexa fluor 647s-phasea

8、nti edll antibody, labeled with alexa fluor 488dna contenthoechst 33342, combined with cellmask blueperklnelmerarw the better细胞周期检测结果i - dna含量分布l178.0e+3integrated dna intensity根据核染色平均荧光强度和总荧 光强度做聚类分析散点图 s-phase细胞核平均荧光强度几乎 没有变化，而面积增大 m-phase 和 early g1phase细胞核面 积变小，荧光强度增大 2n dna (g1-phase)和4n dna (g

9、2- /m-phase)能很明显地区分开f细胞毒性检测perklnelmer0rw the betterl2040-30-1iiiiii-101234log thymidine pmlog nocodazole pm g1-phase ec50 0.12 s-phase- g 2-phase m-phase ec50 0.1 1 gl-pha?ee 36,65 s-phaseec50 37.57 g2-phase m-phase dose-response curves for thymidine (left) and nocodazole (right) effects on cell cy

10、cle 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 (胸腺嗑咤)诱导细胞停滞在glphase nocodazole (诺考达睫)促进细胞进入m-phasef细胞毒性检测结果lcell countperklnelmer0rw the bettercontrola */a 她49%,”“30 川

11、 fqcp 认300 nm tacrine; 5a 100 mm aap % 细胞计数细胞膜透性线粒体数量细胞核增大/皱缩hoechst 33342bobo-3 (not displayed), hoechst 33342mitotracker deep redhoechst 3334220x long wd物镜成像对hepg2细胞进行3种化合物处理(carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone , tacrine and acetaminophen),观察到细胞毒性处理后细胞数量减少，质膜透性增加，线粒体数量增多，细胞核变小log

12、 compound |ec50 ifccp tacrine aap0.572.4 l3 mm cell count is a very sensitive indicator of cytotoxicity (very low ec50 values, compared to other methods)22perklnelmerarw the better细胞毒性检测结果ii -细胞核面积和荧光强度1- - -0050002 11k w &unn50 -3-2-10123456log compound|jfccp tacrineaapecjo |17.2240.8 碓34.2 mm_suyl

13、:23nn细胞损伤后通常会造成细胞核皱缩fccp和tacrine导致细胞核皱缩，同时细胞核荧光强增大aap最初导致细胞核增大，很可能是因为诱导细胞坏死，然后细胞核皱 缩24细胞毒性检测结果iii -线粒体数量perklnelmerarw the better125线粒体在细胞毒作用 早期就受到影响，这 里线粒体功能紊乱主 要表现为线粒体呼吸 作用增强化合物处理24 h后，线 粒体数量增加2000 hiiiiiiiii-3-2-10 i 23456logcompound|fccptacrineaapecq) i5.2117.7n/a细胞毒性检测结果iv-细胞膜透性perklnelmer0rw t

14、he better|logcompound|pm fccp tacrine aapec5() i35.4 pm345.3 pm49.1 mm通过不能透过细胞 膜的bobotm-3染色 定量分析细胞膜受 损伤程度.细胞毒作用后期， 细胞膜完整性降 低,因此根据 bobotm3染色读数 计算的ec50值高于 其它方法得到的26perkinelmekfor the better2012 perkinelmerhuman healthenvironmental health在活细胞内分析nf-kb动 态变化1perklnelmerarw the betterhalotag tmr ligand 染活细

15、胞halotag - tmr liganc (fused to p65 subunit of nf-kb); 30 min post-tnfa addition0 ng/ml tnfa0.5 ng/ml tnfa50 ng/ml tnfa hek293细胞稳定表达nf+b家族带halotag融合标签的p65(p65-ht),通过halotag tmr配体透过细胞膜染活细胞用不同浓度tnfoc刺激nf+b信号用operetta高内涵系统成像，40xlwd物镜荧光宽场21tnfa induces nuclear import of p65nf-kb转位具有时间效应通过harmony高内涵成像和分析

16、软件计算计算nf-kb荧光信号的核质比0.75111-50050100150timeminesjedsao/snoonu)一 0 ng/ml tnfa + 10 ng/ml tnfac刺激30min后，核内p65-ht百分比达到最高值22live cell imaging allows the study of signaling dynamicslperklnelmerarw the better胞内p65分布具有剂量依赖性分析tnfa刺激30 min后，p65.ht分布的剂量依赖性 5 5 50 9 8 1 o oesjedola 0/s nqonshslow 05 1-5958esb-d

17、2a3/sna)onu)now75ill0.75-112 tnfa ng/ml 1010100log tnfa ng/ml ro106-9920um 0,00.85.00.0核内nf+b信号随tnfa浓度的增加而增加(ec5o= 0.5 ng/ml)进入核内的nf-kb随ro106-9920 (稳定抑制nf+b信号)浓度的增加而降低，具有 剂量依赖性通过活细胞的方法检测化合物对nfkb入核或出核的动态变化进行检测23quantitative analysis yields excellent z values 0.7间充质干细胞成脂分化|perkinelmekfor the betterhum

18、an health i environmental healthdata generated in the perkinelmer application lab, hamburg2012 perkinelmerperklnelmer0rw the better间充质干细胞(mscs)成脂分化0 rm insulin1.74 jim insulin10 rm insulin mscs were cultured in adipocyte differentiation medium with different insulin concentrations staining: dapi (nu

19、clei, blue), tritc-phalloidin (actin, yellow), lipidtox (lipid droplets, green) images acquired on the operetta system using 20x high na objective, non-confocal25cellular changes in lipid content and actin cytoskeletonlperklnelmerarw the better按照脂滴含量分类differentiation of mscs into adipocytes100% -90%

20、 -80% -70% -60% -50% -40% -30% -20% -10% -0% -1.74 pm10jiminsulininsulin0 |1minsulinanalysis using the building blocks find nuclei and select cell region in harmony software classification of cells by lipidtox fluorescence in expanded nuclear region26number of adipocytes increases with insulin conce

21、ntration细胞骨架变化的定量分析0 pm insulin10 um insulincalculate texture propertiesser valley 1 px0 |1m insulin10lim insulin(s)en 一q-b) xd l h山s7 6 5 4 3 2 o o o o o oj j j j j j o o o o o o1 o o oj , o o performed texture analysis using harmony software s calculate texture properties building block on an expa

22、nded nuclear regiontexture analysis provides an intensity independent measure of cytoskeletal structure27actin texture changes significantly upon adipogenesisperklnelmer0rw the betterfsmooth granular patchesexamples:looking for different intensity values in neighboring pixels图像的纹理分析36zernike moments

23、: statistics in a single object (e. g. standard deviation)threshold adjacency statistics (tas): intensity relations of neighboring pixelsharalick:intensity relations over larger pixel arrays.perkinelmekfar the betterhuman health i environmental healthphenologictm机器自学 习功能 2009 perkinelmerperklnelmer0

24、rw the better强大的phenologic机器自学习功能f33phenologic应用机器自学习功能可实现： 分析明场图像（label free检测/分 析）轻点鼠标即可快速实现对不同细胞 亚群分类 自动识别不同细胞特征，包括纹理 特征无需过多计算和优化参数设置：所 有的计算和参数优化设置都由columbusfmda-mb-231 pre-migration,. r . , . ( .1 a i oris cell migration assay assay oris 96-well collagen i coated plates with oris cell seeding st

25、oppers; two different cell lines: ht-1080 mda-mb-231incubation for 6 h; remove stoppers;migration for 18 h; fixationstaining with dapi and tritc-phalloidin measurementi 118 h post-migrationmda-mb-231 post-migrationquantify open area remaningfsegmented image: covered (green); free (yellow)teaching te

26、xture class “a” (yellow circles) and texture class b” (green circles)the future: label-free analysis of proliferationcolony segmentation using phenologic machine learningffind texture regionsmodifycoloniesdetected texture regionstrain the machine learning algorithm by clicking into the image!calcula

27、te morphology and intensity propertiesperkinelmekfar the betterhuman health i environmental health基于dpc技术的 细胞追踪harmony 3.5 2009 perkinelmerperklnelmer0rw the betterfdpc数字相差对比度成像33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fperklnelmer0rw the better明场细胞运动轨迹追踪video sequence shows dividing cells correctly track

28、ed on a digital phase contrast imagegreen: track of first generation red: second generation37successful tracking of dividing cells over generations单细胞跟踪 automatically track and analyze the motion of objects works also with dpct=48 hourstracking of hela cells on digital phase image. the track of one

29、cell starting at t=0 (red line) is shownover 48 hours. several cell devisions occur, each color represents a new generation of daughter cells.38successful tracking of dividing cells over generationsperklnelmer0rw the betterf运动轨迹90 100 110生长曲线10 20 30 40 50 60 70 sodpc细胞运动追踪结果33numbercurrentofdisplac

30、etimepoin trackedment xrow_ column plane tcells age(s (?m)current displace menty ?mcurrentspeed (?m/sintensity cellmean -digitalphaseintensitycellnumbercontrast-numberoftrackeddigitalcelloftimepoin trackcells -phase cell area roundne analyzedts perduration gen erar icontrastssfieldstracks|onaccumul

31、ated distanceper trackdisplace ment ?m per trackaverage speed l?m/spj- tfuiknumberof tracesheightterrl?m fnwki ture7713312 1649.786 -4.83242-0.639870.003764s88.68840.888622 723.3874 0.9026621 9.622933 1s403.s6 1.33407857.2353734.177030.00386317900 1799.7667813112 1608.188 -4.983860.0886810.003622579

32、.89350.883073 795.476 0.91s5251 9.836237 1577351 1.52961753.s484831.329420.0036975740 1301.177913265 1653.185 -3.343230.1611650.003607589.13420.892303 724.132 0.9200821 10.51684 16987.59 1.44635964.112338.87050.00398312770 1802.85671013226 1557.196 522981-0.272480.00373601.42560.881812 823.505 0.899

33、1231 9.473144 15128.47 1.42417151.4551231.321230.0035212660 1804.7571113200 1651.874 -2.085771.0279870.002832599.56640.835778 908.2412 0.8621361 8.226003 12898.77 1.45200742.7220726.581440.00344611460 1805.32771213178 1621.532 -5.09446-0.072540.004052570.64340.877574 730.2122 0.907618110.486 16941.0

34、9 1.45016861.2801235.105160.0039528930 1s03.95471313147 1705.868 1.0675473.1283190.003234578.89350.833q47 843.3525 0.91603110.061 16180.07 1.46411555.9693333.795950.0036328360 1804.04771413262 1680.424 -4.02976-0.314650.003673593.97180.887071 754.4873 0.3115711 9.583685 15316.881.4064755.1133332.767

35、890.003768142201804.3971513181 1565.93 -0.707880.4444820.003174573.3630.870023 828.7648 0.9042481 9.08596 14441.15 1.4m57553.6821333.629740.00384110470 1305.313operetta高内涵成像系统dpc明场功能和harmony 3.5软件强大的cell tracking分析功能完美结合，只需简单操作，即可获得细胞分裂或运动的详 细数据，包括分裂次数、生长曲线、运动轨迹、位移、方向、速度等多种 参数结果。lperklnelmer0rw the

36、betterquantifications are accurate300050025000cell countconfluency6050hse,2j0t7 怎 qeml=sn = io5000 cells/well7500 cells/well2500 cells/wello o o o o 4 3 2 1s 8csc802500 cells/well 5000 cells/well 7500 cells/well digital phase contrast fluorescence baseda image analysis of fluorescence vs. digital ph

37、ase images results in similar cell counts and confluency levels at three different cell densities. (image acquisition and analysis as described before.)40fluorescent staining redundant for certain read-outs神经细胞分析模块 powerful neurite analysis left: individual segments (different colors) right: roots (

38、circles), nodes (arrows), and ends (numbers) unique colocalization analysis neurites can be used as search mask for signals colocalized on neurites = essential for axon analysis middle: mask created from neuriteslower: spot identification on neurite (arrow)41perklnelmer4rw the better神经细胞分化分析- marker

39、 expression expression of cell type specific marker sets for neuronal and glia cells analyzed by multi color imaging cells classified by levels of marker expression55classified gfap positive and gfap/tui1 positive cells fperklnelmer4rw the betterneuronal and glia cells, differentiated from embryonal

40、 stem cells, stained fortujl (red) and gfap (green)ytoplasmic regions for quantification of cell markersperklnelmerarw the betterfxenon lampdichroic mirror wheeloptional confocal light path, pinhole discy operetta硬件配置专门为hcs设计的光路高qe14bitccd适应hcs定量需求自动化物镜与滤光片管理系统 300w高能氤灯 740nm led明场光源emission filter

41、wheel三种成像光路瓢远红外高速自动对焦声三，w长工作距离和高数值孔径物镜可供选择 适应各种不同微孔板的成像要求片全光谱覆盖的光路八个激发光滤光片转轮,(360-640 nm)八个条形码自动识别的发射光滤光片位置 15个不同波长的滤光片可供选择45维护窗口fperklnelmer0rw the better齐全的物镜 2x.100x物镜可供选择有pe-scan功能，方便用户扫描特定孔板区域条形码自动识别全自动物镜工作距离判定与保护，防止物镜受损高数值孔径物镜符合高质量图像要求长工作物镜适应各种不同规格的微孔板47物镜数值孔径工作距离2x long wd0.086.2 mm10x long wd0.310 mmlox high na0.43.21 mm20x long wd0.457.8 mm20x high na0.750.71 mm40x long wd0.64.0 mm40x high na0.950.29 mm60x long wd0.72.31 mm60x high na0.90.31 mmloox long wd0.851.2 mm软件直观易用perkinelmerarw