Analyzing Single Molecule Localization Microscopy Data Using Cubic Splines

The resolution of super-resolution microscopy based on single molecule localization is in part determined by the accuracy of the localization algorithm. In most published approaches to date this localization is done by fitting an analytical function that approximates the point spread function (PSF) of the microscope. However, particularly for localization in 3D, analytical functions such as a Gaussian, which are computationally inexpensive, may not accurately capture the PSF shape leading to reduced fitting accuracy. On the other hand, analytical functions that can accurately capture the PSF shape, such as those based on pupil functions, can be computationally expensive. Here we investigate the use of cubic splines as an alternative fitting approach. We demonstrate that cubic splines can capture the shape of any PSF with high accuracy and that they can be used for fitting the PSF with only a 2–3x increase in computation time as compared to Gaussian fitting. We provide an open-source software package that measures the PSF of any microscope and uses the measured PSF to perform 3D single molecule localization microscopy analysis with reasonable accuracy and speed.