DETERMINATION OF 3-DIMENSIONAL IMAGING PROPERTIES OF A LIGHT-MICROSCOPE SYSTEM - PARTIAL CONFOCAL BEHAVIOR IN EPIFLUORESCENCE MICROSCOPY
DETERMINATION OF 3-DIMENSIONAL IMAGING PROPERTIES OF A LIGHT-MICROSCOPE SYSTEM - PARTIAL CONFOCAL BEHAVIOR IN EPIFLUORESCENCE MICROSCOPY
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DOI：
10.1016/s0006-3495(90)82534-0
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发表时间：
1990-02-01
影响因子：
3.4
通讯作者：
AGARD, DA
中科院分区：
文献类型：
--
作者：
HIRAOKA, Y;SEDAT, JW;AGARD, DA
我们已经确定了三维图像形成特性的一个荧光显微镜用于获得非常高分辨率的三维图像的生物结构的图像处理方法。三维显微数据被收集为在不同焦平面上记录的一系列二维图像。这些图像不仅包含焦平面周围区域的聚焦信息，还包含标本其余部分的失焦贡献。一旦显微镜系统的成像特性被表征，强大的图像处理方法可以用来消除失焦信息和纠正图像畸变。虽然对无像差显微镜系统的行为的理论计算是可用的，但在用于生物观察的条件下，真实透镜的性质往往远不理想。因此，我们直接确定了在与生物观察相关的条件下，荧光显微镜的成像特性。在电荷耦合器件图像检测器上记录了点物体（荧光涂层微球）的通焦系列。从这些图像中，导出了三维点扩散函数及其傅里叶变换，即光学传递函数。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。这种差异可以解释为显微镜系统的缺陷，非理想的观察条件，和部分共聚焦效应发现发生与荧光照明。了解显微镜系统的光学行为表明如何优化标本制备，数据收集和处理协议，以获得显著改善的图像。
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.