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共焦和数字光片成像

光学切片–两种方式

光学成像仪器可以放大微小的物体,放大远处的恒星,并揭示肉眼看不见的细节。但是众所周知,它存在一个烦人的问题:景深有限。我们的眼透镜(光学成像仪器)也有同样的麻烦,但是我们的大脑会在信号到达有意识的认知之前,巧妙地删除所有未聚焦的信息。如果图像保留在照片上,则无法使用。通过优化参数,我们可以增加焦点的深度,但随后通常会丢失分辨率以及z位置的信息。

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如果可以只记录聚焦的特征并测量相对的z位置,则可以解决聚焦深度问题:记录一堆图像后,我们可以重建整个样本,而没有模糊的部分并量化对象。所有这三个维度。

1.垂直方式-共聚焦

Most commonly, we have 共聚焦 microscopy [1] 在提及光学切片时请牢记。共焦原理已无缝集成到标准型光学显微镜中(尽管技术集成需要进行很多修改)。与普通的光学显微镜不同,真正的共聚焦成像需要对单个光斑进行尽可能小的照明。光斑的直径由波动光学系统决定,并在d处达到衍射极限的最小值 ≈ λ/ NA。详细的强度分布由点扩展函数描述。同时,探测器还必须感应到尽可能小的斑点。由于光路是对称的,因此相同的数学运算适用于点状检测器的感测分布。通过将针孔孔径引入到检测路径中的中间像平面中来实现点检测器。在样本中,照明和检测的焦点必须重合-因此"confocal"显微镜检查。如图所示 如图1所示,此针孔的效果是光学去除了所有不是从焦平面发出的光线。它的功能是z方向上的空间滤波器。

由于光学刀一次只能在一个点上工作,因此该点必须从上到下成行移动才能生成图像。强度被同步转换为数字信息,并存储在计算机上的帧存储中。从该电子存储器中,图像显示在普通监视器上。由于必须逐点顺序构造图像,因此只有在某些影响帧格式和可接受的信噪比的限制下,才可能实现高帧速率。高度透明的光路,例如Leica SP 检测器以及非常高效的传感器(例如混合检测器(HyDs))有助于提高高帧率性能。同时,线路频率高达12,000 Hz are implemented in 共聚焦 microscopes, compared to ca 15,000 PAL标准电视中的Hz线路频率。在适当的条件下,帧速率最高可达每秒500个是可能的。尽管如此,要获得足够高的信噪比图像,帧频通常不会超过10 frames per second.

与扫描过程有关的另一个问题是,焦平面上方和下方的样品区域会暴露于大量的照明能量中,这对图像没有帮助,但可能导致荧光染料的光物理降解。由于荧光染料也在焦平面上方吸收光,因此当深入样品中时,荧光强度会越来越暗。这可以通过自动增益自适应来补偿,但是以信噪比为代价。

The positive aspects of true 共聚焦 imaging are 日 e high resolution (high-NA lenses are easily applied), 日 e fact 日 at 日 e image is inherently homogeneous in 日 e focal plane and 日 at standard preparations are easily and effortlessly analyzed with 共聚焦 microscopes. Confocal microscopes are based on standard coaxial light microscopes, and all standard microscopy methods and contrast modes are accessible to 日 e microscopist by a simple switch. 它也是其他类型的扫描显微镜的基础,例如多光子激发,高次谐波产生显微镜,相干反斯托克斯拉曼散射显微镜和超分辨率技术 STED (受激发射耗竭显微镜)。

2.水平方向–灯片

Although 日 e 共聚焦 microscope is 日 e gold standard for optical sectioning, 日 e first microscope to perform 日 is task was introduced half a century before. The "Spalt-Ultramikroskop" [2] 使用聚焦在狭缝光圈中的明亮光源(弧光灯或太阳)。第二个透镜(通常是可重复使用的物镜)将该狭缝垂直于显微镜的光轴投射到样本中。这种设计有效地创建了大约两微米厚的片状照明。最初的应用与亚分辨率粒子有关,例如在彩色玻璃中分散金。事实证明,它适用于各种胶体样品和混浊介质,在化学和医学研究中通常会对其进行分析。这款早期的光片显微镜仅限于观察小至5的微小颗粒 纳米,导致照明的球形散射,在显微镜的垂直光路中可观察到作为衍射图样。结果,它可视化了不可分辨的粒子,称为"Ultramikronen", which are "超出显微镜的分辨率". The name "ultramicroscope" would suggest a superresolution type of design in 日 e first place, but it was made plain by 日 e authors 日 at 日 e 超显微镜 was clearly limited by optical diffraction.


图2:Spalt-Ultramikroskop中光路的示意图。激发光由物镜1通过狭缝(Spalt)正交聚焦到样品中。该发射由物镜2收集并且可以由照相机记录为光学部分。

后来,开发出绕过精密狭缝的设计,并采用了筒形透镜来产生垂直于光轴的光 [3]。然后将这种安排用于荧光固定样品。事实证明,这种薄板显微镜对厚样品的快速实时成像特别有利,因为漂白现象不那么明显,并且由数码相机并行进行图像记录共焦和数字光片成像 如Huisken所示 [6]。在z方向上的吸收不是问题,因为显微术师正在聚焦的z位置上的所有位置的照明均相等。但是,发射光必须像普通显微镜一样通过样品,这可能会使厚样品中的图像在某种程度上劣化。仍然在横向上有阴影效果:首先在照明光进入样品的一侧吸收光,因此荧光在相反的一侧变暗。伏伊 [3] 通过旋转样本并收集不同方向的图像来补偿这种影响。多特 [7] 从两个相对的侧面照亮样品,以补偿光片的吸收。凯勒(Keller)无需使用投影的狭缝孔径或柱面透镜, [4] 扫描垂直于观察轴的细激光束。这个方案叫做"数字扫描的光片".

3. The conjunction

Both paradigms have been introduced in biomedical research and routine, serving for many new insights and understandings of biological concepts and diseases, and of 日 e construction and function of cellular components. In terms of instrumentation, 日 e two ways are significantly different, 日 e orthogonal illumination was treated as a separate part of 日 e system and combined with a coaxial microscope in order to perform 日 e task. The 共聚焦 microscope uses a beam-scanning system to create twodimensional images, 日 e digitally scanned light sheet uses beam scanning to create areas from lines. This immediately raises 日 e question of whether it would be possible to combine both strategies in one single system. 徕卡微系统 introduces such a combination in 日 e new Leica TCS SP8 DLS 光片显微镜 [5].

徕卡 TCS SP8 DLS is based on an ordinary 共聚焦 microscope, a coaxial scanning device 日 at performs optical sectioning by spatially filtering 日 e out-of-focus light by virtue of a detection pinhole. Images are generated by means of galvanometric scanning mirrors 日 at move 日 e illumination spot in 日 e two lateral directions. To convert such a system into a digital 光片显微镜, one must make 日 e illumination beam pass 日 rough 日 e sample perpendicular to 日 e optical axis. By introducing a small mirror at 日 e position of 日 e focal plane, just outside 日 e observed field, 日 e required orthogonal illumination is achieved. Such a deflecting mirror is mechanically connected to 日 e observation lens to ensure 日 e illuminated z-position is always in 日 e focus of 日 e image-generating optics, while 日 e image is projected onto a video camera 日 at converts 日 e intensities into a digital image.

A single position of 日 e illumination beam would not cause a two-dimensional image, but only a single line. One can make use of one of 日 e lateral scanning modes inherently implemented in a 共聚焦 microscope to scan 日 e illumination line in 日 e perpendicular direction, yielding 日 e required two-dimensional plane.

If a second mirror is introduced on 日 e opposite side, it is possible to illuminate 日 e sample from two sides 日 rough 日 e same lens – just by employing 日 e second axis of 日 e beam-scanning device of 日 e 共聚焦 microscope. This fulfills 日 e need to compensate for absorption-caused shadowing. The final image is 日 en again constructed from 日 e two images with different illumination directions.

For imaging in 共聚焦 mode, 日 e z-drive mechanism has to be positioned so 日 at 日 e focus of 日 e illumination beam coincides with 日 e feature 日 at is to be imaged. The scan process 日 en covers just 日 e inside of 日 e field of view, which is 日 e common way to operate a 共聚焦 microscope. To switch to light-sheet acquisition, 日 e focus has to be moved by about 日 e distance of 日 e mirror to 日 e optical axis. In 日 is case, 日 e plane-generating part of 日 e illumination beam will fall into 日 e center of 日 e observation field. The scanning device needs to point to 日 e edge of 日 e field of view in order to hit 日 e mirror.

This combination does not only reduce instrumentation cost, as two different instruments are merged into one, but it also allows 日 e combination of classical 共聚焦 and digital light-sheet microscopy. As a consequence, 日 e same system allows any sample to be explored with 日 e technique 日 at is most suitable to address 日 e current research question. It also enables a variety of new application opportunities since 日 e 共聚焦 path could be used for photo-manipulations with subsequent gentle light-sheet imaging of 日 e induced effects.