第十七章流式细胞仪分析技术及应用课件_2

第十七章 流式细胞仪分析技术及应用 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 第一节 概述 一、工作原理 二、散射光的测定 三、荧光测量 四、细胞分选原理 第二节 数据的显示与分析 一、参数 二、数据显示方式 三、设门分析技术 第三节 流式细胞仪免疫分析的技术要求 一、免疫检测样品制备 二、免疫分析中常用的荧光染料与标记染色 三、免疫胶乳颗粒的应用 四、流式细胞免疫学技术的质量控制 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 第四节 流式细胞术在免疫学检查中的应用 一、淋巴细胞及其亚群的分析 二、淋巴细胞功能分析 三、淋巴造血系统分化抗原及白血病免疫分型 四、肿瘤耐药相关蛋白分析 五、AIDS病检测中的应用 六、自身免疫性疾病相关HLA抗原分析 七、移植免疫中的应用 思考题 小结 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 流式细胞术(flow cytometry, FCM)是以流式 细胞仪为检测手段的一项能快速、精确的对 单个细胞理化特性进行多参数定量分析和分 选的新技术。 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 流式细胞术最大的特点是能在保持细胞及 细胞器或微粒的结构及功能不被破坏的状态下 ，通过荧光探针的协助，从分子水平上获取多 种信号对细胞进行定量分析或纯化分选。 细胞不被破坏，测量快速、大量、准确、灵敏、定量 流式细胞术的特点 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 流式细胞仪是测量染色细胞标记物荧 光强度的细胞分析仪，是在单个细胞分析 和分选基础上发展起来的对细胞的物理或 化学性质（如大小、内部结构、DNA、RNA 、蛋白质、抗原等）进行快速测量并可分 类收集的高技术。 第一节 概述 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 细胞组成细胞功能 大小细胞表面/胞浆/核-特异性抗原 粒度细胞活性 DNA, RNA含量胞内细胞因子 蛋白质含量激素结合位点 钙离子, PH值, 膜电位酶活性 流式细胞仪常检测的细胞特性 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 采用激光作为激发光源，保证其具有更好的单色性与激发 效率； 利用荧光染料与单克隆抗体技术结合的标记技术，保证检 测的灵敏度和特异性； 用计算机系统对流动的单细胞悬液中单个细胞的多个参数 信号进行数据处理分析，保证了检测速度与统计分析精确 性。 一、工作原理 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. （1） 液流系统 （2） 光学系统 （3） 数据处理系统 1.流式细胞仪的基本结构： Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 由样本和鞘液组成 待测细胞 单个细胞的悬液 荧光染料标记的 单抗对其染色 受清洁气体压力 从样品管进 入流动室形成样本流 鞘液：辅助样本流被正常检测的基质液。主要作用是 包裹样本流的周围，保持样本流中细胞处于喷嘴中心位 置，防止其靠近孔壁而阻塞喷孔。 （1）液流系统 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 喷嘴 Fluorescence signals Focused laser beam 鞘液 液流系统示意图 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. FCM的液流系统（如 何形成单个细胞流） 样本管 鞘液管 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 激光光源：气冷式氩离子激光器 分色反光镜：反射长/短波长，通过短/长 波长 光束成形器：两十字交叉放置的透镜 透镜组：形成平行光，除去室内光 滤片：长通、短通、带通 光电倍增管：FS, SS（散射光）， FL1, FL2, FL3, FL4（荧光） （2）光学系统 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. Flow Tip Laser SS and FL Detector FS Detector 光学系统示意图 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 主要由计算机及其软件组成 （3）数据处理系统 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 基本工作原理 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 单细胞液柱 已标记的单细 胞悬液和鞘液 硅化管 流动室喷嘴 荧光检测系统和 散射光感受系统 收集光信号 荧光染料被 激发发光 光电倍增管 脉冲信号 计算机系统 分析结果 放大 垂直相交 形成稳态水平激光与之 基本过程 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 区别别流式细细胞仪仪显显微镜镜 光源激光自然光、灯光 对对象细细胞、生物粒子细细胞、组织组织 等 承载载工具鞘液及流动动室载载玻片 检测检测 信号光学信号形态态及染色 放大方式PMT、放大电电路目镜镜物镜镜、光学放大 统计统计计计算机，5000人工，200 结结果多参数，综综合分析简单简单 ，单单参数 流式细胞仪与显微镜的区别 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 细胞在液柱中与激光束相交时 向周围360立体角方向散射的光线 信号，它的强弱与细胞的大小、形 状、胞内颗粒折射等有关，主要分 为前向散射光和侧向散射光。 二、散射光的测定 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 前向散射光（forward scatter, FS）：激光束照射细胞时 ，光以相对轴较小角度(0.510)向前方散射的讯号用于检测 细胞等粒子的表面属性，信号强弱与细胞体积大小成正比。 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. FALS Sensor Laser 前向散射光示意图 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 侧向散射光（side scatter, SS）：激光束照射细 胞时，光以90角散射的讯号，用于检测细胞内部 结构属性。 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. FALS Sensor 90LS Sensor Laser 侧向散射光示意图 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 测得的FS与 SS信号通过计算机处理 ，可得到FS-SS图，由 此可仅用散射光信号对 未染色的活细胞进行分 析或分选。 此为血细胞分类 的基本原理，但不能分 析表面分子。 淋巴细胞 单核细胞 中性粒细胞 光散射测量最有效用途：从非均一群体中鉴别出某些亚群 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 荧光信号由被检细胞上标记的特异性荧光染料受激 发后产生，发射的荧光波长与激发光波长不同。 每种荧光染料会产生特定波长的荧光和颜色，通过 波长选择通透性滤片，可将不同波长的散射光和荧光 信号区分开，送入不同的光电倍增管。 选择不同的单抗及染料就可同时测定一个细胞上的 多个不同特征。 线性放大器和对数放大器 三、荧光测量 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 荧光染料的特性 激发波长(EXCITING) 发射波长(EMISSION) Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 荧光补偿 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 通过流式细胞仪进行细胞分选 主要是在对具有某种特征的细胞需 进一步培养和研究时进行的。 四、细胞分选原理 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 细胞悬液形成液流柱 流动室振动 液流断裂成液滴 空白液滴含细胞的液滴 弃去 偏转落入收集器 压电晶体产生机械振动 不充电充电 （一）分选基本原理 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 分选速度：单位时间内分选的细胞数量。与悬 液中细胞的含量成正比。 分选纯度：分选出的目的细胞占所有收获细胞 的百分率。 分选收获率：实际收获的分选细胞与设定通过 测量点的分选细胞之间的比率。与纯度成反比。 分选得率：从一群体细胞悬液中分辨出目的细 胞的总量，再经分选后得到目的细胞的实际得率。 与分选速度成反比。 （二）分选的技术要求 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 参数：FS,SS,FL 数据显示方式 （单参数直方图 、双 参数散点图 、二维等高图 、假三维等 高图 、三参数散点图 ） 设门分析技术 第二节 数据的显示与分析 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. FS：反映颗粒的大小 SS：反映颗粒的内部结构复杂程度 FL：反映颗粒被染上的荧光数量多少 一、 参数 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 单参数直方图 双参数直方图:点图 二维等高图 假三维等高图 三参数直方图 多参数分析 直分析方图 设门分析:REGION和GATE设置 二、数据显示方式 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 由一维参数(散射光或荧光)与颗粒计数 (COUNT)构成，反映同样散射光或荧光强 度的颗粒数量的多少。 （一）单参数直方图 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 单参数直方图 细胞相对数 量 信道(channel ) Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 双参数直方图：纵轴和横轴分别代表被测 量细胞的两个测量参数，根据这两个参数就 可以确定细胞在图上的表达位置。 双参数信号通常采用对数信号，最常用的 是点密图，在图中，每个点代表一个细胞， 点图利用颗粒密度反映同样散射光或荧光强 度的颗粒数量的多少。 （二）双参数直方图 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 1.双参数直方图点图 绿色荧光强度 红色荧光强 度 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 双参数直方图点图 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 2. 二维等高图 由类似地图上的等高线组成，其本质也是 双参数直方图。 等高图上每一条连续曲线上具有相同的细 胞相对或绝对数，即“等高”。 曲线层次越高(越里面的线) 所代表的细胞 数愈多。 等高线越密集则表示细胞数变化率越大。 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 二维等高图 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 3. 假三维等高图 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. （三）三参数直方图 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 多参数分析：当细胞标记了多色荧光，被激 发光激发后，得到的荧光信号和散射光信号 可根据需要进行组合分析。 （四）流式细胞仪的多参数分析 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. Gate设置：指在某一张选定参数的直 方图上，根据该图的细胞群分布选定其 中想要分析的特定细胞群，并要求该样 本所有其他参数组合的直方图只体现这 群细胞的分布情况。 根据门的形状又分为了线性门、矩形门、圆 形门、多边形门、任意形状门和十字门。 三、设门分析技术 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. A 淋巴细胞 B 单核细胞 C 中性粒细胞 A、B、C均 为任意门 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 线性门 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. Region设置: 区阈（region, R）与门(gate, G ) 是两个 相关的概念，区阈可与门对应，但是也可 以包含于门。 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. D1 CD4+/CD3- D2 CD4+/CD3+ D3 CD4- /CD3- D4 CD4- /CD3+ 如十字门分析时，起 就可以由四个区域构 成，即 G=D1+D2+D3+D4。 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 样本制备 标记染色 液相芯片技术 质量控制 第四节 流式细胞仪免疫分析的技术要求 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 外周血淋巴细胞样品的制备 分离单个核细胞 培养细胞的样品制备 蛋白酶消化 机械吹打 使贴壁细胞脱落 洗涤 尼龙网过滤 单细胞悬液 一、免疫检测样品制备 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 新鲜实体组织单细胞悬液的制备 机械法 酶处理法 化学试剂处理法 表面活性剂处理法 单细胞悬液的保存 深低温保存法（一年） 乙醇或甲醇保存法（2周） 甲醛或多聚甲醛保存法（2月） Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 适用条件: 有较高的量子产额和消光系数 对488nm的激发光波长有较强的吸收 发射光波长与激发光波长间有较大的波长差 易与标记单抗结合而不影响抗体的特异性 二、常用的荧光染料与标记染色 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. FITC Texas red PE.PC.APC PEcy5 FL1 FL2 FL3 FL4 激光 细 胞 悬 液 异硫氰酸荧光素 得州红 能量传递复合染料 藻胆蛋白类 （一）几种常见的荧光染料 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 名称染料激发 波长 荧光颜 色 溶解性对PH敏感 性 特点 异硫氰酸荧 光素 FITC488绿 525 易敏感易溶于水，与抗体结 合不影响特异性 得州红Texas red 568红 615 不易不敏感稳定，偶联后量子产 额低 藻红蛋白PE488橙 575 易不敏感具较多发光基团，消 光系数和量子产额高 藻青蛋白PC488 别藻青蛋白APC633红 670 能量传递复 合染料 PEcy5488红 670 易不敏感减少交叉，成本高 常用的几类荧光染料 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 用化学法将两种不同激发波长的染 料结合在一起，在488nm激发光照射下，通过 一个荧光染料被激发后产生的发射波长再激 发另一荧光染料产生荧光信号，从而检测到 该特定荧光信号。 能量传递复合染料 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. PE CY5 CY5 CY5 488nm 575nm 670nm 红色荧光 花青苷5 藻红蛋白 能量传递复合染料机制 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile .Created with Aspose.Slides for .NET 3.5 Client Profile . Copyright 2004