荧光显微镜的基本使用方法

激发光源
• 高压汞灯拥有313, 337, 365, 405, 436, 546, and 577 纳米的峰值谱。高压 Xenon灯有连续的可见光光 谱范围, 光源强度比较平均。
汞灯光谱
激发滤色块
• 具有多套激发滤色块：激发滤色镜（Excitation Filter），分 光镜（Dichromatic Mirror），发射滤色镜（Emission Filter）。
（儿茶酚胺、5－羟色胺、组胺等）在甲醛作用下 呈不同颜色的荧光。 • 组织内含有的奎宁、四环素等药物也呈现一定的 荧光。 • 嗜中性粒细胞中脱氢酶辅酶呈现一定的荧光。
荧光素 DAPI AMCA Hoechst 33258 FITC Acridine Orange Acridine Yellow CY3 TRITC Propidium Iodide
BX51 在镜基上装有3个滤色 片（ND6，ND25，LBD）， 另外，还可以选装第四个。 镜基侧面的拉杆可以很容易 地插入/取出滤色镜。
Specimen color fading can be delayed by reducing the excitation light intensity with ND filters. Use the ND filters as far as they do not hinder observations. Insert the ND filters (U25ND6 and/or U-25ND25) with the marked side facing toward the observer.
激发波长 372 350 365 490 490 470 552 541 530
发射波长 456 450 465 520 590 550 565 572 615
Fra Baidu bibliotek
荧光显微镜的优点和用途
优点： 检出能力高 对细胞的刺激小 能进行多重染色 用途： 物体构造的观察 荧光的有无、色调比较进行物质判别 发荧光量的测定对物质定性、定量分析
激发滤色块
六孔激发块转盘
多用途型BX-RFA(适合多种科研要求)及通用型BX-URA2。可同时使用6种荧光 激发块， “轻拨式”激发块转换功能，使不同荧光激发之间的迅速转换非常方
便，并可消除震动，有利于复染样品的荧光观察。激发块标识在暗室环境中发 光，在暗室中也能很容易地选择需要的荧光激发块。另外，根据需要还可以选 择6孔滤色片滑块(U-RSL6/U-RSL6EM)安放激发滤色片和发射滤色片进行荧光 观察。
实验原理
实验原理
荧光显微镜的光路及主要部件
光源 激发光挡片 激发滤色镜 分色镜
Microscopes with an upright-style frame are capable of producing fluorescence illumination either through episcopic or diascopic optical pathways, although the latter is rarely used today. Epi-illuminators usually consist of a mercury or xenon lamphouse coupled to a vertical illuminator that is positioned above the main frame in a separate assembly. The microscope nosepiece and transmitted light components (diascopic illuminator, condenser, field diaphragm, filters, etc.) are built into the main frame, while fluorescence components are housed in the vertical illuminator. These illuminators often contain a revolving or sliding turret that houses four to six "cubes" that contain a mixture of interference filters including a barrier filter, dichroic mirror, and an excitation filter. As illustrated above, light emitted from the lamp positioned in the episcopic lamphouse passes through a collector lens and then the field and aperture diaphragms before entering the first interference filter in the cube set, the emission filter. This light is then directed through the objective and onto the specimen by a special dichroic mirror that reflects certain wavelengths while passing others. Secondary fluorescence, emitted by fluorophores residing in the specimen, travels back through the objective and the dichroic mirror before passing through a barrier filter and into the microscope eyepieces or camera system.
细胞生物学实验
荧光显微镜的基本使用方法
实验目的
1. 了解荧光显微镜的基本原理； 2. 掌握荧光显微镜的基本结构和 使用注意事项；
自发性荧光物质
• 神经细胞和心肌细胞等内的脂褐素呈棕黄色荧光。 • 肝贮脂细胞和视网膜色素上皮细胞内的维生素A呈
绿色荧光。 • 某些神经内分泌细胞和神经纤维内的单胺类物质