Tahmasbi Amir, Ward E Sally, Ober Raimund J
Opt Express. 2015 Mar 23;23(6):7630-52. doi: 10.1364/OE.23.007630.
Fluorescence microscopy is a photon-limited imaging modality that allows the study of subcellular objects and processes with high specificity. The best possible accuracy (standard deviation) with which an object of interest can be localized when imaged using a fluorescence microscope is typically calculated using the Cramér-Rao lower bound, that is, the inverse of the Fisher information. However, the current approach for the calculation of the best possible localization accuracy relies on an analytical expression for the image of the object. This can pose practical challenges since it is often difficult to find appropriate analytical models for the images of general objects. In this study, we instead develop an approach that directly uses an experimentally collected image set to calculate the best possible localization accuracy for a general subcellular object. In this approach, we fit splines, i.e. smoothly connected piecewise polynomials, to the experimentally collected image set to provide a continuous model of the object, which can then be used for the calculation of the best possible localization accuracy. Due to its practical importance, we investigate in detail the application of the proposed approach in single molecule fluorescence microscopy. In this case, the object of interest is a point source and, therefore, the acquired image set pertains to an experimental point spread function.
荧光显微镜是一种受光子限制的成像方式，它能够以高特异性研究亚细胞物体和过程。使用荧光显微镜成像时，感兴趣物体定位的最佳可能精度（标准差）通常使用克拉美罗下界来计算，即费希尔信息的倒数。然而，当前计算最佳可能定位精度的方法依赖于物体图像的解析表达式。这可能带来实际挑战，因为通常很难为一般物体的图像找到合适的解析模型。在本研究中，我们转而开发一种直接使用实验收集的图像集来计算一般亚细胞物体最佳可能定位精度的方法。在这种方法中，我们将样条（即平滑连接的分段多项式）拟合到实验收集的图像集，以提供物体的连续模型，然后可用于计算最佳可能定位精度。由于其实际重要性，我们详细研究了所提出方法在单分子荧光显微镜中的应用。在这种情况下，感兴趣物体是一个点源，因此，采集的图像集与实验点扩散函数相关。
