Thomann D, Rines D R, Sorger P K, Danuser G
Bio Micro Metrics Group, Laboratory for Biomechanics, Swiss Federal Institute of Technology, Wagistrasse 4, CH - 8952 Schlieren, Switzerland.
J Microsc. 2002 Oct;208(Pt 1):49-64. doi: 10.1046/j.1365-2818.2002.01066.x.
In this paper, we describe an algorithmic framework for the automatic detection of diffraction-limited fluorescent spots in 3D optical images at a separation below the Rayleigh limit, i.e. with super-resolution. We demonstrate the potential of super-resolution detection by tracking fluorescently tagged chromosomes during mitosis in budding yeast. Our biological objective is to identify and analyse the proteins responsible for the generation of tensile force during chromosome segregation. Dynamic measurements in living cells are made possible by green fluorescent protein (GFP)-tagging chromosomes and spindle pole bodies to generate cells carrying four fluorescent spots, and observe the motion of the spots over time using 3D-fluorescence microscopy. The central problem in spot detection arises with the partial or complete overlap of spots when tagged objects are separated by distances below the resolution of the optics. To detect multiple spots under these conditions, a set of candidate mixture models is built, and the best candidate is selected from the set based on chi2-statistics of the residuals in least-square fits of the models to the image data. Even with images having a signal-to-noise ratio (SNR) as low as 5-10, we are able to increase the resolution two-fold below the Rayleigh limit. In images with a SNR of 5-10, the accuracy with which isolated tags can be localized is less than 5 nm. For two tags separated by less than the Rayleigh limit, the localization accuracy is found to be between 10 and 20 nm, depending on the effective point-to-point distance. This indicates the intimate relationship between resolution and localization accuracy.
在本文中，我们描述了一种算法框架，用于自动检测三维光学图像中瑞利极限以下间距的衍射极限荧光斑点，即具有超分辨率。我们通过追踪芽殖酵母有丝分裂过程中荧光标记的染色体，展示了超分辨率检测的潜力。我们的生物学目标是识别和分析在染色体分离过程中产生拉力的蛋白质。通过用绿色荧光蛋白（GFP）标记染色体和纺锤极体，使活细胞动态测量成为可能，从而产生携带四个荧光斑点的细胞，并使用三维荧光显微镜观察这些斑点随时间的运动。当标记物体之间的距离低于光学分辨率时，斑点检测的核心问题在于斑点的部分或完全重叠。为了在这些条件下检测多个斑点，构建了一组候选混合模型，并基于模型与图像数据的最小二乘拟合中残差的卡方统计量，从该组中选择最佳候选模型。即使对于信噪比（SNR）低至5 - 10的图像，我们也能够在瑞利极限以下将分辨率提高两倍。在信噪比为5 - 10的图像中，孤立标签的定位精度小于5纳米。对于间距小于瑞利极限的两个标签，根据有效点对点距离，定位精度在10到20纳米之间。这表明分辨率与定位精度之间存在密切关系。
