概述
荧光是指物质吸收某个波长的光，然后释放出另一个波长的光的现象。受到激发跃迁到一个较高的不稳定能量状态的电子，在回到其基础状态时释放出一个光子，就产生了荧光。导致激发的光，也就是使电子跃迁到较高能量状态的光，比释放的荧光有更短的波长和更高的能量。释放的荧光有较长的波长、较低的能量、以及不同的颜色。
荧光显微镜技术结合了光学显微镜的放大特征和荧光技术。荧光技术能够激发荧光基团这种荧光化学物质，并能检测到来自荧光基团的发射光。有了荧光显微镜技术，科学家可以观察组织内特定细胞类型的位置或者细胞内某种分子的位置。
荧光显微镜的原理和组成
荧光显微镜的主要部件和传统的光学显微镜大致相同。而两个主要的不同点是光源的类型和使用特殊的滤光元件。荧光显微镜需要非常强的光源，如氙光灯或您这里看到的水银灯。水银灯发出的光要比大多数白炽灯亮10到100倍，光的波长也很宽，从紫外到红外。这种高强光源是荧光显微镜最危险的部分，因为直视这种没有过滤的光会严重伤害您的视网膜，错误地操作灯泡会导致其爆炸。
荧光显微镜技术的原理很简单。来自水银灯的光通过激发滤光片，后者会选择透过激发光的波长。过滤后的光被一种称为分色镜的特殊镜片反射到样品上，分色镜只会反射具有激发波长的光。反射的光通过物镜到达聚焦好的荧光样品上。然后来自样品的发射光又反过来通过物镜，这时图像信号被放大，然后再通过分色镜。发射光会被阻挡滤光片过滤，阻挡滤光片只选择透过发射波长的光，会过滤掉来自水银灯以及其他显微镜组件反射的杂光。最后，经过过滤的发射荧光被送到探测器，图像在那里会被数字化，或者被传输到显微镜目镜用于肉眼观察。激发滤光片、分色镜以及阻挡滤光片能够被组装在一起成为一个部件，称为滤片块。观察样品时，可以调换不同的滤片块来改变激发光波长，还可用一系列的光阑来调节激发光的强度。
操作荧光显微镜技术时，荧光基团和显微镜一样的重要，用于成像的荧光基团类型决定了使用的激发光波长和可检测的发射光波长。激发光波长包含了可被荧光基团吸收并将其跃迁到激发状态的小部分能量。被激发后，电子重新跃迁到原来的低能量状态，就可能会产生范围较广的发射光谱。吸收峰或称激发曲线与发射曲线峰值之间的差别就是斯托克斯位移。这种位移的距离越大，区分两种不同波长的光就越容易。另外，任何重合的光谱都需要利用滤片块来去除，这样可以降低背景并增加图像的质量。荧光基团如果过长时间暴露于激发状态会导致光漂白，即荧光的减弱或消失。为降低光漂白，可在载玻片切片上加入抗光漂白封片液，然后用指甲油封闭边缘。当不进行成像时，切片应当避光保存。
荧光显微镜成像
开始荧光成像时，要先打开氙光灯或者水银灯，预热大约15分钟以得到持续稳定的光源。再将您的样品放到载物台上并固定好。然后，打开显微镜的白光灯源。使用最低倍数的物镜，通过调节粗调和细调聚焦旋钮对样品进行聚焦。再使用载物台调节旋钮来找到想观察的区域。下一步是关掉白光灯源，以及任何不需要的室内灯光以减少背景。根据所用成像的染料选择正确的滤片块，打开光栅照射您的样品。最后，调节细调聚焦旋钮并将输出光转到成像照相机上。您可能会需要调节曝光时间来观察不同的荧光基团或不同的荧光染料。
但是，当比较同一染料染色的不同样品时，使用相同的曝光时间非常重要。当使用多种染料染色成像同一样品时，则需要调节滤片块来观察相应的荧光基团并记录新的图像。样品中的每种染料都被成像之后，可将多个图像叠加和融合。
应用
许多不同类型的实验都会用到荧光显微镜和不同类型的荧光基团。荧光显微镜技术最普遍的应用之一是使用结合了荧光化合物的抗体来识别蛋白质并进行成像。这里，钩端螺旋体表面蛋白的抗体，被一个结合了Alexa Fluor荧光素的二抗识别，激发时产生绿色荧光。
另一个使用荧光技术特色的方法是将编码荧光蛋白如绿色荧光蛋白GFP的基因编码，插入到生物组织的DNA当中。GFP基因来源于水母，可在特定诱导条件下由培养细胞表达合成，也可在某些特定类型细胞如肿瘤细胞中表达，正如您在这里看到的图像中的发光的部分。
另一个荧光成像的应用是荧光斑点显微镜技术。这种技术使用的是荧光标记的超级大分子聚合体，如这里的F-肌动蛋白网络，用来研究这类重要的骨架蛋白的运动和动态转换。
还有一种先进的技术叫荧光漂白恢复技术FRAP，它特意在样品的一个小区域进行光漂白，来检测荧光标记分子扩散回到光漂白区域的速率。
结语
您刚观看的是JoVE关于荧光显微镜的介绍。本短片中，我们学习了荧光的概念，荧光显微镜与光学显微镜的区别，如何利用显微镜进行荧光成像。我们还学习了一些使用荧光的基础和先进的应用。感谢观看。不要忘记，漂白使您的牙齿好看，但对您的样品可不好。
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