Kozubek M
Faculty of Informatics, Masaryk University, Brno, Czech Republic.
Microsc Res Tech. 2001 Apr 15;53(2):157-66. doi: 10.1002/jemt.1080.
The aim of this article is to compare experimental resolution under different conditions with theoretical resolution predicted using electromagnetic diffraction theory. Imaging properties of fluorescent beads of three different diameters (0.1 microm, 0.2 microm, and 0.5 microm) as well as imaging properties of four different fluorescence-stained DNA targets (ABL gene, BCR gene, centromere 6, and centromere 17) are studied. It is shown how the dependence of the resolution on object size varies with wavelength (520 nm versus 580 nm), type of microscopy (wide-field, confocal using Nipkow disk, confocal laser scanning) and basic image processing steps (median and gaussian filters). Furthermore, specimen influence on the resolution was studied (the influence of embedding medium, coverglass thickness, and depth below the coverglass). Both lateral and axial resolutions are presented. The results clearly show that real objects are far from being points and that experimental resolution is often much worse than the theoretical one. Although the article concentrates on fluorescence imaging using high NA objectives, similar dependence can also be expected for other optical arrangements.
本文的目的是将不同条件下的实验分辨率与使用电磁衍射理论预测的理论分辨率进行比较。研究了三种不同直径（0.1微米、0.2微米和0.5微米）的荧光微珠的成像特性以及四种不同荧光染色的DNA靶点（ABL基因、BCR基因、6号着丝粒和17号着丝粒）的成像特性。展示了分辨率对物体大小的依赖性如何随波长（520纳米与580纳米）、显微镜类型（宽视场、使用尼普科夫盘的共聚焦、共聚焦激光扫描）和基本图像处理步骤（中值和高斯滤波器）而变化。此外，还研究了样本对分辨率的影响（包埋介质、盖玻片厚度和盖玻片下方深度的影响）。给出了横向分辨率和轴向分辨率。结果清楚地表明，真实物体远非点状物，并且实验分辨率通常比理论分辨率差得多。尽管本文主要关注使用高数值孔径物镜的荧光成像，但对于其他光学装置也可预期有类似的依赖性。
