Randall Division of Cell and Molecular Biophysics, New Hunt's House, Guy's Campus, King's College London, SE1 1UL (United Kingdom).
Chemphyschem. 2014 Mar 17;15(4):677-86. doi: 10.1002/cphc.201300756. Epub 2014 Jan 31.
Localization microscopy vastly improves the resolution achieved by fluorescence microscopy by fitting the positions of individual fluorophores. We examine the reconstructions produced by different fitting algorithms for instances of fixed pattern noise--systematic tendencies to alter estimated emitter positions according to their subpixel location in a way that does not reflect the ground truth structure. We show that while not readily visible at standard empirical signal strengths, fixed pattern noise can occur when performing sub-pixel fitting, and that its degree varies according to the algorithm used and the relative size of the pixels compared to the point spread function. For pixel sizes in the range 80-170 nm, this results in variations in accuracy of the order of 2-4 nm-comparatively small for many applications, but non-negligible in scenarios where very high accuracy is sought.
定位显微镜通过拟合单个荧光团的位置，极大地提高了荧光显微镜的分辨率。我们研究了不同拟合算法在固定模式噪声情况下产生的重建，固定模式噪声是一种系统趋势，根据荧光团在亚像素位置的情况改变估计的发射位置，而这种改变方式与真实结构无关。我们表明，虽然在标准经验信号强度下不易察觉，但在进行亚像素拟合时可能会出现固定模式噪声，其程度取决于所使用的算法以及像素大小与点扩散函数的相对大小。对于 80-170nm 范围内的像素大小，这会导致精度变化约 2-4nm——对于许多应用来说相对较小，但在需要非常高准确性的情况下不容忽视。
