光学显微镜是一种被研究人员在许多领域使用，用来放大观察样品的仪器，可将样品放大到原来大小的1000倍。最简单的光学显微镜包含一个用来放大样品的干净镜头和一个照射样品的光源。但大多数光学显微镜都要更复杂，在显微镜内部和其物镜目镜里，还包含了很多严格控制了尺寸大小的高精度镜头。本短片将描述光学显微镜的主要组件，并详细讲解它们的应用和功能。还会介绍放大、聚焦以及分辨率这些基本原理。基本的光学显微镜操作，要先将光源照射在样品上，并保证光源的强度、方向及形状合适，这样才能产生最佳质量的图像。然后需要将样品合适地放大并聚焦，来观察感兴趣的区域。光学显微镜有很多实际的应用，包括观察染色或者未染色的细胞或组织，仔细研究样品的微小细节，甚至可以在手术过程中放大感兴趣的区域以帮助在微米级别上的复杂操作。
概述
光学显微镜是用于放大观察研究样品的仪器。它是非常有价值的研究工具，可使科学研究人员将物体放大到原来尺寸的1000倍来进行观察。正如您将要看到的，尽管光学显微镜运用的是一些最基本的原理，但它在观察实验室样品方面却有着无数的应用。
光学显微镜的基本原理
如其名字所指，光学显微镜需要一个光源，其发出的光被聚焦透镜聚焦后照射到样品上。照亮样品的光到达一个称为物镜的镜头，它会生成一个放大的倒置图像。接目镜，也称目镜，会进一步放大图像，图像然后被眼睛接收。其他的光学元件也可以加入到光通道中来修正图像，这样使眼睛看到的样品是正确的方向。使用多种镜头的显微镜，正如这里您见到的这个，被称为复式显微镜。在复式显微镜中，总的放大倍数等于物镜的倍数乘以目镜的倍数。所以40倍的物镜和10倍的目镜组合，总的放大倍数是400倍。可以使用目镜网线，也就是投射到图像上一个刻度，来帮助估计被观察物体的大小。当使用高倍数放大时，目镜网线上的刻度代表的距离比使用低倍数放大时要小。
除了放大倍数，显微镜的另外一个光学属性是分辨率。分辨率是指在显微镜下，两个独立物体之间能被分辨的最短距离。随着分辨率的提高，这些图像上的特征越来越清晰，也就是它们之间能被观察到的最短距离减小了。
光学显微镜的主要组件
光学显微镜的主要组件包括：物镜、目镜、载物台和样品夹架、光源、视场光圈、聚光镜和聚光孔以及粗调和细调聚焦旋钮。
显微镜的放大倍数和分辨率主要取决于物镜。它们固定在换镜旋座上，因此当它们转换时，焦平面保持不变，这种特性称为平行聚焦。物镜上可以标有放大倍数、数值孔径NA、物镜浸没介质的类型、固定样品的盖玻片厚度以及工作距离，也就是指镜头顶端到样品聚焦平面的距离。数值孔径，又被定义为N.A.,用于衡量显微镜物镜接收光的能力。高数值孔径的物镜可接收斜射过来的光，而低数值孔径的物镜仅能接收较直射的光。若已知光波波长，物镜的分辨率可通过数值孔径得出。
光源、视场光圈、聚光孔以及聚光镜用于产生和调节投射到样品上的光。光源通常是低压卤素灯泡，可以通过调节改变光的强度。然后光通过不同的滤光片进入视场光圈，后者会调节样品被照射的面积大小。接下来是聚光镜，它会将光聚集到样品上。根据所使用物镜的类型，可通过聚光镜来调节照射在样品上的圆锥形光柱。
操作光学显微镜
使用光学显微镜前，先要将含有想观察区域的样品放到显微镜载物台上，置于物镜之下，并用载物台夹子固定。接下来，打开光源，选择最低倍数的物镜。
然后，使用起始的粗调旋钮在Z轴方向上下移动物镜进行聚焦。然后旋转细调旋钮进行精细聚焦。注意不要让物镜撞击到载玻片或者载物台，否则会损坏镜头。
然后，一边调节旋钮使载玻片在X轴和Y轴方向前后左右移动，一边从目镜观察寻找想观察的区域。当放大倍数从小换到大时，观察区域的面积会大幅度减小。因此调换较高倍数物镜前，先在低倍数物镜下将感兴趣的区域移到观察视野中心，会大幅度提高找到所需样品的几率。一旦样品在低倍物镜下被定位和聚焦，就可转到高倍物镜下进行成像。优化光源时，首先调节视场光圈使得光圈恰好位于视野的外围，然后调节聚焦光圈，使得它与物镜的数值孔径一致。
最后，使用细调旋钮调节再次进行聚焦。现在您就可以开始对样品进行拍照了。
应用
光学显微镜能够观察多种样品，针对用途的不同，有不同构造的显微镜可以使用。您这里看到的是一个研究人员正准备使用手术显微镜进行工作。这种显微镜通常悬挂固定在一个活动杆上，并且是立体显微镜，是指它们可以将光同时传给观察者和一个固定在显微镜上的照相机。这台手术显微镜正在用于小鼠的一个肾脏移植手术。
在这个剪辑中，您看到的是一个研究人员正在通过解剖显微镜观察选出最佳的果蝇幼虫，以作进一步解剖暴露体表的肌肉，研究神经和肌肉的连接点。这里您看到的是一个为显微注射准备的倒置复式显微镜，其物镜位于载物台下面。这种实验操作，又称为体细胞核转移技术，是制备转基因动物和创造克隆体的一种重要方法。
结语
您刚观看的是JoVE对于光学显微镜的介绍。在本视频中我们回顾了：什么是显微镜和它是怎样工作的，它的组件，如何调节使用，和如何获取高质量的图像。感谢观看！
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