吴立君博士徕卡显微系统南京大学共聚焦显微镜生物学相关荧光样品制备共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014植物细胞动物细胞共聚焦显微镜生物学相关荧光样品制备Lijun Wu Leica / Shanghai 23/6/2014共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014MitoTracker LysoTrackerAnti-Calnexin TubulinGM-130Phalloidin Hoechst 33258EEA1DAPIPIOrganelle markersActinPIEREarly Endosome荧光样品染色•免疫荧光细胞化学法•荧光探针•荧光蛋白基因转染用于体内标记的荧光探针•量子点法共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014组织标本主要取之活组织检查标本、手术切除标本、动物模型标本以及尸体解剖标本。

前三作用是沉淀蛋白质和糖，对组织穿透性很强，保存抗原的免疫活性较好。

但醇类对低分子蛋白质、多肽及胞浆内蛋白质的保存效果较差，解决的办法是和其他试为免疫组织化学染色中最常用的一种切片方法。

其最突出的优点是能够比较完好地保存多种抗原的免疫活性，尤其是细胞表面抗原更应该采用冰冻切片。

新鲜的组织及已经固定的组织共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014组织和细胞的自发性荧光检测1、动物组织一般呈现弱淡兰色荧光，其中弹性纤维的荧光较强；2、红细胞血红蛋白中的卟啉呈红色荧光，但在正常情况下，卟啉和铁离子相结合，铁离子有抑制荧光作用，所以红细胞不发荧光。

如在血液中加酸使铁离子游离，或缺铁性贫血，红细胞可呈红色荧光；3、细胞内的脂褐素呈棕黄色的自发荧光，例如心肌细胞核两侧的肌浆内，随着年龄的增长或在某些疾病的情况下，可见较大量的棕黄色脂褐素荧光颗粒；4、某些肿瘤细胞在紫外或蓝光激发可呈现明显的自发荧光，可作原位肿瘤的早期诊断；5、维生素A呈绿色的自发性荧光。

如大鼠食入维生素A后，在肝细胞、Kuffer细胞、肾上腺束状带细胞、肺和肾的间质细胞、卵巢间质细胞和黄体细胞等的胞质内均可见到维生素A的绿色荧光；6、某些药物也可呈现一定颜色荧光，如奎宁为绿色荧光，四环素为黄色荧光。

四环素能与骨基质中的钙结合，利用这一特性，可以用四环素饲养动物以观察新骨质的形成。

另外，四环素和癌细胞有较大的亲合力，能在恶性肿瘤内形成黄色的荧光灶；7、有机体的单胺神经原、消化道和呼吸道粘膜内神经内分泌细胞中的内源性生物胺—儿茶酚胺、5-羟色胺和组织胺等与某些醛类物质在一定条件下可发生缩合反应而呈现荧光。

醛和儿茶酚胺（多巴胺、去甲肾上腺素和肾上腺素）缩合可产生亮黄绿色荧光，和5-羟色胺及组织胺缩合后呈现黄色荧光。

该方法可显示脑干中单胺神经元胞体内、神经纤维中、消化道呼吸道粘膜的内分泌细胞中的去甲肾上腺素和5-羟色胺，以及交感神经末梢内含量低至5X10-10g的去甲肾上腺素。

共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014免疫荧光组化技术是根据抗原—抗体反应的原理，小鼠耳部真皮层免疫荧光染色绿色：示CCL21（淋巴细胞区划因子）红色a：示Perlecan（基底膜蛋白多糖）红色b：示Coll IV（IV型胶原）青色：TRITC-dextr（TRITC-葡聚糖）30线粒体探针MitotrackerCellular LocalizationCalculation for Fura-IndicatorsD.J. Linden, Purkinje neuronFura-2共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014Calcium Green-1笼锁化合物探针(解笼锁，uncage)笼锁化合物全称为光不稳定笼锁化合物，是人工使用掩蔽基团修饰生物活性分子而合成的无荧光前体化合物。

当该物质被强光照射后，两基团之间的共价键解离而释放出活性分子，这个过程称为解笼锁。

荧光染料是否发射荧光或荧光的强弱，主要决定于该染料的分子结构。

同时，与它所处的环境及其状态也有密切关系。

如染色液的pH值、浓度和染色时的温度以及缓冲液离子浓度等。

绿色荧光蛋白小鼠•GFP可以作为唯一的工具，在活细胞内监测基因表达和蛋白质定位。

•由于GFP由生物发光蛋白组成，水母蛋白（Ca共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014of fluorescence microscopy changed dramatically withof fluorescent proteins in the1950s.The starting pointdetection of the jellyfish Aequorea victoria green fluorescentby Osamo Shimomura.Hundreds of GFP mutants later,荧光蛋白The FP beachR.Tsien 2009 Nobel lectureCyan (CFP)Y66W Green (GFP)S65T Yellow (YFP)S65G, T203YRed (d.s.RFP)共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014A: EGFP-fluorescence in endosomes ofVero cellsB: Immunostaining against endosomalmarker EEA1, cy5-conjugated secondaryabLijun Wu Leica / Shanghai 23/6/2014共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014法，磷酸钙法，电穿孔法，脂质体法各有利弊，其主要原理及应用特点见下表：共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014GFP´s as Sensor Molecules共聚焦显微镜生物学荧光新技术吴立君博士徕卡显微系统南京大学23/6/2014共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014No Color Correction in IR HC PL IRAPO 40x/1.10 W CORR 405 nm488 nm633 nm5 µm Leica HC PL APO 40x/1.30 Oil CS2STEDColocalization FCSLive Cell Fast Imaging OPO + Ti:Sa Electrophysiology Deep Tissue Imaging CARS UV ExcitationPhase Contrast ＆DICMitoTracker LysoTrackerAnti-Calnexin TubulinGM-130Phalloidin Hoechst 33258EEA1DAPIOrganelle markersActinPIEREarly EndosomeSynaptogyrinsynaptic vesicles are complicatedTakamori et al. 2006 Cell 共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014荧光样品染色•免疫荧光细胞化学法•荧光探针•荧光蛋白基因转染用于体内标记的荧光探针•量子点法共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014•GFP可以作为唯一的工具，在活细胞内监测基因表达和蛋白质定位。

•由于GFP由生物发光蛋白组成，水母蛋白（Ca 绿色荧光蛋白小鼠荧光蛋白R.Tsien 2009 Nobel lecture The FP beachLijun Wu Leica / Shanghai 23/6/2014共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014QuantitationMeasure averagefluorescence intensitiesin regions of interests.BleachTotalBg 共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014PA-GFP and HcRed cotransfected NRK cells Courtesy: P. Lenart, Ellenberg group, EMBL, Heidelberg, Germany 光诱导变色荧光蛋白KEADE目前已经从珊瑚虫和海葵中提取和开发出了许多有应用潜力的光学指示剂。

其中一个非常重要的是Kaede蛋白，受紫外激发后这种蛋白会产生从绿色到红色的光转化。

光转化后，红/绿比会显著增加约2000倍（红增、绿减）。

这一类具有独特颜色转换特性的光学指示剂会成为亚细胞器乃至整个细胞光学标记的最佳选择。

共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014HEp-2 cells (Hemmerich, Jena, Germany)(aCBP/p300, aRNA-polymerase II, DAPI)Zebrafish (Bastmeyer, Konstanz, Germany) (Two different neuronal markers)HEK cells (Böhmer, Jena, Germany) (free GFP, YFP_SHP1 fusion)??Provided the general theoretical framework for FRET applications.In 1946he proposed a mathematical law for dependence of fluorescence decay of donor (D)on the concentration of an acceptor (A),assuming a dipole-dipole interaction in solution (Naturwissenschaften 6:166-175)founded onFöster Resonance 1912non-radiationless 共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014Lijun Wu Leica / Shanghai 23/6/2014共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014FRET in action 共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014Preconditions for FRET AnalysisSpecific staining of the molecule (protein) of interestFluorochromeAny of various fluorescentsubstances used influorescence microscopy to共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014 FRET WIZARD共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014道的一种直(multicolor BiFC)不合体的形成，间产目前，蛋白蛋白较究工作上共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014Figure 6. From: BIMOLECULAR FLUORESCENCE COMPLEMENTATION (BiFC) ANALYSIS AS A PROBE OF PROTEIN INTERACTIONS IN LIVING CELLS. Concurrent visualization of multiple protein complexes using multicolor fluorescence complementation analysis. (a) Two alternative interaction partners, A and B, are fused to fragments of different fluorescent proteins (YN155 and CN155 respectively). These fusions are cells with a shared interaction partner, Z, fused to a complementary fragment (CC155). Complexes formed by A-YN155 and Z-CC155 can be distinguished from complexes formed by B-CN155 and Z-CC155 based on their fluorescence spectra. (b) Schematic representation of the visualization of multiple protein complexes in the same cell (A-YN155-Z-CC155, cytoplasmic and perinuclear; B-CN155-Z-CC155, nuclear and Tom K. Kerppola. Annu Rev Biophys. Annu Rev Biophys;37:465-487.共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014TCS SP8 SMD Series• A platform that integrates the confocal system TCS SP8 with SMD specific components from Picoquant•Used for FLIM, FLIM-FRET, FCS, FCCS, gated FCS, FLCS, and FLCCSLeica TCS SP8SMD specific componentsconfidentialLifetime dependency on local ion concentration or pHConventional confocal intensity image Fluorescence lifetime image of cells expressing CFP-YFPCourtesy: G. Hams, University of WürzburgBiological heterogeneity of FRET cells (CFP-YFP fusion), Courtesy: G. Hams, University of WürzburgFLIM image of scale insect antenna (non-stained):Image was reconstructed from a 3-dimensional FLIM-z-stack,Courtesy of Kees Jalink共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014较短的IRF 保证可靠的FLIM-FRET 数据Sample: fixed cells with GFP or GFP-mCherry FRET construct, excitation @ 900 nm, detection @ 500-550nm, FRET efficiency = 31%Courtesy: Corentin Spriet, Imaging Center Lille, Franceconfidential共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014白激光高图像反差的光闸技术从470-670nm的自由可调激发波长（1nm步进）高达同时8条可调谱线，3万亿个独特组合›2D激发－发射扫描的综合光谱图像›应用于FLIM和gSTED的脉冲激发光源›利用自由光谱开发新的图像策略共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014Picosecond Laser at 1060 nmSeed SourcePumpPower AmplifierSupercontinuum FiberSupercontinuum GenerationSupercontinuum Fiber (cross section)WLL -Supercontinuum Laser ConceptTCSPC -T ime C orrelated S ingle P hoton C ounting+ good time resolution!+ high sensitivity!0 13 15 18 20 28 40 100 2200 s 100 s 190 s250 s共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/2014SP8X FLIM: Excitation at variable wavelength1.9 ns3.0 nsABCSample: fixed cells with triple staining:GalNacT2_GFP (golgi), LAMP-546 (endosomes), Calnexin 594 (ER).A: 486 nm, B: 542 nm, C: 594 nmSample: courtesy of Matthias Weiss, Cellular Biophysics Group,Bioquant, Heidelberg, Germany.The FLIM measurements at different excitation wavelengths (A: 486 nm, B: 542 nm, C: 594 nm show specific fluorescence lifetimes of the three 共聚焦显微镜生物学相关荧光样品制备Dr Lijun Wu Leica / Shanghai 23/6/20141.9 ns3.0 ns ABCλ2 plot: The three fluorescence peaksrepresent the three labels: GalNacT2_GFP(golgi), LAMP-546 (endosomes), Calnexin594 (ER).。