Evaluating performance in three-dimensional fluorescence microscopy.
Evaluating performance in three-dimensional fluorescence microscopy.
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评估三维荧光显微镜的性能。
DOI：
10.1111/j.1365-2818.2007.01861.x
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发表时间：
2007-12
影响因子：
2
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中科院分区：
文献类型：
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在生物荧光显微镜中，图像对比度通常会因样本的焦点区域外产生的高背景而降低。这种背景可以大大减少或消除厚标本显微镜的几种模式，包括技术，如3-D去卷积和共焦。在原则上或实践中，这些方法中哪一种更“好”，引起了很大的兴趣和一些混乱。这里报告的实验的动机是建立一些粗略的指导方针，为给定的生物标本选择最合适的显微镜方法。该方法是比较光子收集的效率，图像的对比度和信噪比实现的不同的方法在等效照明，使用的标本，其中的量的焦点外的背景是可调的范围内遇到的生物样品。我们比较了点扫描共聚焦，旋转盘共聚焦和宽场/解卷积（WFD）显微镜，发现在焦点信号的比例的焦点外的背景，可以用来预测哪种方法的显微镜将提供最有用的图像。我们还发现，净荧光产量的测量精度是非常低于预期的所有模式的显微镜。我们的分析能够清晰，定量地描绘出不同成像模式的适当使用相对于离焦背景与对焦信号的比率，并定义了三种最常见的成像模式的有用范围的上限。
In biological fluorescence microscopy, image contrast is often degraded by a high background arising from out of focus regions of the specimen. This background can be greatly reduced or eliminated by several modes of thick specimen microscopy, including techniques such as 3-D deconvolution and confocal. There has been a great deal of interest and some confusion about which of these methods is ‘better’, in principle or in practice. The motivation for the experiments reported here is to establish some rough guidelines for choosing the most appropriate method of microscopy for a given biological specimen. The approach is to compare the efficiency of photon collection, the image contrast and the signal-to-noise ratio achieved by the different methods at equivalent illumination, using a specimen in which the amount of out of focus background is adjustable over the range encountered with biological samples. We compared spot scanning confocal, spinning disk confocal and wide-field/deconvolution (WFD) microscopes and find that the ratio of out of focus background to in-focus signal can be used to predict which method of microscopy will provide the most useful image. We also find that the precision of measurements of net fluorescence yield is very much lower than expected for all modes of microscopy. Our analysis enabled a clear, quantitative delineation of the appropriate use of different imaging modes relative to the ratio of out-of-focus background to in-focus signal, and defines an upper limit to the useful range of the three most common modes of imaging.