Hiraoka Y, Sedat J W, Agard D A
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
Biophys J. 1990 Feb;57(2):325-33. doi: 10.1016/S0006-3495(90)82534-0.
We have determined the three-dimensional image-forming properties of an epifluorescence microscope for use in obtaining very high resolution three-dimensional images of biological structures by image processing methods. Three-dimensional microscopic data is collected as a series of two-dimensional images recorded at different focal planes. Each of these images contains not only in-focus information from the region around the focal plane, but also out-of-focus contributions from the remainder of the specimen. Once the imaging properties of the microscope system are characterized, powerful image processing methods can be utilized to remove the out-of-focus information and to correct for image distortions. Although theoretical calculations for the behavior of an aberration-free microscope system are available, the properties of real lenses under the conditions used for biological observation are often far from an ideal. For this reason, we have directly determined the image-forming properties of an epifluorescence microscope under conditions relevant to biological observations. Through-focus series of a point object (fluorescently-coated microspheres) were recorded on a charge-coupled device image detector. From these images, the three-dimensional point spread function and its Fourier transform, the optical transfer function, were derived. There were significant differences between the experimental results and the theoretical models which have important implications for image processing. The discrepancies can be explained by imperfections of the microscope system, nonideal observation conditions, and partial confocal effects found to occur with epifluorescence illumination. Understanding the optical behavior of the microscope system has indicated how to optimize specimen preparation, data collection, and processing protocols to obtain significantly improved images.
我们已经确定了落射荧光显微镜的三维成像特性，该显微镜用于通过图像处理方法获取生物结构的超高分辨率三维图像。三维显微数据作为在不同焦平面记录的一系列二维图像进行收集。这些图像中的每一幅不仅包含来自焦平面周围区域的聚焦信息，还包含来自标本其余部分的离焦贡献。一旦表征了显微镜系统的成像特性，就可以利用强大的图像处理方法去除离焦信息并校正图像畸变。尽管有无像差显微镜系统行为的理论计算，但用于生物观察的实际透镜在这些条件下的特性往往远非理想。因此，我们直接确定了落射荧光显微镜在与生物观察相关的条件下的成像特性。在电荷耦合器件图像探测器上记录了点物体（荧光涂层微球）的透焦系列图像。从这些图像中，推导出了三维点扩散函数及其傅里叶变换——光学传递函数。实验结果与理论模型之间存在显著差异，这对图像处理具有重要意义。这些差异可以通过显微镜系统的不完善、非理想的观察条件以及落射荧光照明中发现的部分共焦效应来解释。了解显微镜系统的光学行为表明了如何优化标本制备、数据收集和处理方案，以获得显著改善的图像。
