3-DIMENSIONAL IMAGING BY CONFOCAL SCANNING FLUORESCENCE MICROSCOPY
3-DIMENSIONAL IMAGING BY CONFOCAL SCANNING FLUORESCENCE MICROSCOPY
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DOI：
10.1111/j.1749-6632.1986.tb34549.x
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发表时间：
1986-12-31
影响因子：
5.2
通讯作者：
NANNINGA, N
中科院分区：
文献类型：
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作者：
BRAKENHOFF, GJ;VANDERVOORT, HTM;NANNINGA, N
追求共聚焦显微镜的决定源于对电子显微镜在亚微米范围内获得生物标本活形态可靠数据的可能性的不满。这种可能性受到各种样品制备方法对材料表观结构的影响的强烈限制。制备所需的步骤（化学固定、脱水和薄切片）各有各的效果。例如，已经观察到细菌的体积收缩高达50%。“由此产生的对成像技术的兴趣比标准光学显微镜具有更好的分辨率，但在自然环境中仍然可以观察到标本，这使我们在光学显微镜中应用了共聚焦原理。”如果采用高数值孔径（NA= 1.3-1.4）的光学器件，就有望超越标准光学显微镜的分辨率限制。我们小组在透射共聚焦显微镜中实际演示了这一事实3，结果在633 nm波长处观察到196 nm的点响应，在442和325 nm波长处观察到130-140 nm的点响应。共聚焦原理也适用于荧光显微镜，实际上，由于荧光光的不相干性，可能会产生更高的分辨率。但是，即使在实际考虑排除这些分辨率的条件下（见下文），在这种模式中仍然存在所谓的切片效应，通过这种效应，样品中离焦层的荧光贡献被阻止了对图像形成的贡献。这种贡献导致正常荧光的有效对比度大大降低。在这种类型的显微镜中产生数据的串行方式，加上切片效果，使共聚焦扫描激光显微镜（CSLM）特别适合耦合到计算机系统。这样一种仪器就可以用相对简单的方法对生物标本进行高分辨率的三维研究。在描述了扫描显微镜（光学部分，仪器控制，计算机系统，使用的处理算法）之后，我们介绍了空间点响应的测量和三维成像在生物学中的一些应用。最后，我们将讨论目前正在开发的各种形式的扫描显微镜的优点和局限性。
The decision to pursue confocal microscopy was born from dissatisfaction with the possibility of electron microscopy to acquire reliable data about the live morphology of biological specimens in the submicron range. This possibility is strongly restricted by the way various specimen-preparation methods affect the apparent structure in the material. The required steps in the preparation (chemical fixation, dehydration and thin sectioning) each introduce their own type of effect. For instance, a volume shrinkage of bacteria has been observed with values up to 50%.'The resulting interest in imaging techniques with a better resolution than standard light microscopy, but where the specimen still could be observed live in its natural environment, led us to the application in light microscopy of the confocal principle. With this approach, which has been used before in acoustic microscopy: the resolution limitations of standard light microscopy could be expected to be surpassed if optics of high numerical aperture (NA= 1.3-1.4) were used. The actual demonstration of this fact in transmission confocal microscopy by our group3 resulted in observed point responses of 196 nm at a wavelength of 633 nm and 130-140 nm at wavelengths of 442 and 325 nm. The confocal principle is also applicable in fluorescence microscopy where, actually due to the incoherence of the fluorescence light, an even higher resolution may re~ ult.~ But even under conditions where practical considerations exclude these resolutions (see below), there still remains the so-called sectioning effect in this mode by which fluorescence contributions from off-focus layers in the specimen are prevented from contributing to the image formation. Such contributions lead in normal fluorescence to a strong reduction of the available contrast.The serial way in which the data are produced in this type of microscope, together with the sectioning effect, makes the confocal scanning laser microscope (CSLM) particularly suitable for coupling to a computer system. Thus an apparatus results which permits three-dimensional studies of biological specimens at high resolution with relatively simple instr~ mentation.~-~ After a description of the scanning microscope (optical part, instrument control, computer system, processing algorithms used) we present a measurement of the spatial point response and a number of applications of three-dimensional imaging in biology. Finally, we will discuss some of the merits and limitations of the various forms of scanning microscopy presently under development.