简单地说,共聚焦显微镜是一种显微镜技术,它获取通过光学处理 从焦点平面 去除失焦光后得到的图像。所得图像减轻了模糊,提高了对比度且具有更高的分辨率,而这些正是共聚焦用途的主要目标。由于每个图像都在具体的焦点平面显示荧光样品,因此可以通过采集多个图像并按顺序 更改 z 轴 焦点平面位置来构造三维图像。此类图像可用于准确确定样品的三维定位。
图 1:共聚焦显微镜和标准宽视野显微镜的图像比较。
图 2:用共聚焦显微镜采集的三维图像示例。
自此,共聚焦显微镜的原理以易于理解的方式得以解释,展示了如何去除焦点平面以外的光。
要测量荧光,就需要向样品发射激发光。在本节,我们将沿着激发光的路径来描述共聚焦显微镜的原理,包括它产生荧光的位置。
在共聚焦显微镜中,激光光束主要用作激发光源。激光光束是具有良好直线性能的点光源,因此其焦点是通过物镜在焦点平面形成的光斑。*1 与一次激发整个样品区域的常规宽视野荧光显微镜相比,共聚焦显微镜一次仅激发焦点平面上的一个点。
*1:物镜放大率、NA 和激发波长的组合方式决定了可以聚焦的最小光斑大小。 NA 越高且波长越短,光斑尺寸就越小,分辨率也越高。
图 3:概念图:激光光束聚焦在焦点平面上。
观察光路穿过空气是有难度的。因此,我们在物镜上方放置了一块荧光材料。当来自物镜的激发光穿过荧光材料块时,光路中的荧光材料就会向各个方向发射荧光。通过观察荧光来跟踪激发光的路径,如图 4 所示。 光束在焦点平面形成一个光斑,另一方面,当使用常规宽视野显微镜时,激发光仍会分散在整个样品上。
图 4:激光器激发光与标准宽视野显微镜激发光的图像比较。
如上所述,激光光束聚焦到焦点平面上的一个点,形成光斑;然而,在 z 轴方向上,激光光束由物镜以该过程对应的宽度进行收敛和发散。当然,激光在焦点平面上下移动,遇到荧光材料时,就会发出荧光。在图 5 中,我们可以看到荧光块中焦点平面的上方和下方在发射荧光。
这种失焦荧光也出现在真实样品中,是导致图像模糊的原因。在共聚焦显微镜中,发射到焦点平面外的荧光受阻,因而不会到达探测器。这样就可以仅使用焦点平面的光来生成图像。
图 5:该图显示了激光光束如何收敛,然后向焦点平面外发散。
共聚焦显微镜如何消除焦点平面外的荧光?
通过跟踪从焦点平面及其上下方产生后直接辐照到探测器的荧光的光路,可以解释共聚焦显微镜的原理。
图 6:概念图:检测从焦点平面发射的荧光。
物镜焦点平面产生的荧光从物镜穿过成像透镜,并在探测器上形成聚焦图像。将针孔放置在成像系统的这个位置。
在这种共聚焦光学场景中,通常将以下三个物体放置在接合点:
A. 光源(更准确地说,是光源点)
B. 物镜的焦点平面
C. 探测器前面的针孔
如果点 A、B、C 构成共轭位置关系,则其中任何一个点发出的光都将聚焦在另外两个点上。A 点发射的光会同时聚焦于 B 点和 C 点。C 点发射的光会同时聚焦于 A 点和 B 点,反之亦然。
那么在共聚焦光学中,失焦光源发射的荧光如何在光路中传播?
图 7:概念图:消除发射到焦点平面外的荧光的技术。
光源发射的光聚焦在物镜焦点平面和探测器前面的针孔中。
然后,在物镜焦点平面处新生成的荧光聚焦在探测器前面的针孔中。从光源发射的激发光被带通滤波器排斥(阻挡),因此在实际的共聚焦系统中(在图 7 左侧的蓝色圆圈中)检测不到激发光。
如图 7 所示,焦点平面上方发射的荧光聚焦在针孔上方,并且由于光在针孔处扩散而基本上被阻挡,无法到达探测器。
其次,如蓝色圆圈中所示,在焦点平面底部发射的荧光聚焦在针孔的底部,因此在很大程度上被阻挡,无法到达探测器。*2
这样,只有从焦点平面发射的荧光可以到达探测器。
*2:由于样品上的光斑大小随物镜而变化,针孔位置的荧光光斑大小也会随之变化。通常会调整共聚焦显微镜的针孔大小以匹配物镜。在我们的共聚焦装置 MAICO 中,可以根据物镜选择三种不同的针孔大小(小、中、大)。
激发光在光路中存在荧光材料的任何位置都能产生荧光。然而,通过仅选择从共轭位置发射到针孔中的荧光,可以获得仅源自焦点的图像。这称为光学切片图像。共聚焦显微镜的主要优点是能够从厚样品中获得该光学切片。
在截至目前的说明中,激发光斑都固定在单个点上,但在实际操作中,会采用光学扫描装置在二维(x、y)方向移动光斑,以产生单个共聚焦荧光图像。在我们的共聚焦装置 MAICO 中,这种光学扫描采用 MEMS 振镜执行。*3
图 8 展示了 MEMS 振镜在光路中改变角度以及激发点在样品上的相关横向移动的过程。图 9 展示了连续光学扫描样品平面的概念图。
*3 有关 MEMS 振镜的更多信息,请查看 特点。
与宽视野激发相比,扫描激发点具有另一个优势。邻近的荧光分子不被激发,并且不太可能产生杂散荧光。
图 8*:概念图:MEMS 振镜的移动和激发光斑的移动。
* 这与实际图像采集操作不同。
图 9:概念图:激发光斑对焦点平面进行平面扫描
PMT(光电倍增管)是光电检测器,根据入射到受光表面上的荧光量随时间输出电信号,但没有位置信息。
另一方面,光学扫描装置在 XY 方向上扫描固定位置达到固定的时间。
PMT 的信号输出会在任意给定时间从信号位置重构。也就是说,信号强度的时间信息被转换成空间信息,并且通过将辉度信息分配给图像的 XY 位置来生成图像。
图 10:示例:PMT 的信号输出随时间转换为二维图像。
如介绍中所述,通过共聚焦测量获得的光学截面图像反映特定焦点平面的样本,因此通常可以构造三维图像。
图 11*:聚焦的激光光斑随物镜的垂直移动而移动,并且采集的图像也会相应地改变。
图 11 显示,通过垂直移动物镜,样品上的焦点位置也会垂直移动。因此,共聚焦图像位置也随着焦点的垂直移动而变化。通过对在图 11 中获取的各个高度位置产生的各个共聚焦图像进行三维重构,可以构建图 2 中的三维图像。
*在图 11 左边的图像中,没有扫描光斑,但在实际测量中,会以扫描设备在 XY 平面扫描光斑。焦点依次上下移动。
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