3-D PSF fitting for fluorescence microscopy: implementation and localization application.
Super-resolution localization microscopy methods such as PALM and STORM have been shown to provide imaging with resolutions up to a few tens of nanometers while using relatively simple setups. Biplane PALM has extended the PALM technique to three-dimensions, by simultaneously using two imaging planes, with different focal depths. A key aspect in achieving good axial localization results is the alignment of the two planes. Currently available approaches assume that misaligned planes only result in scaling and rotation of the PSF pattern. We show in this work that this does not necessarily hold true, especially in the presence of refractive index mismatch between the different optical layers. Instead, we suggest a calibration algorithm that relies on a realistic PSF model and finds the affine transform that relates the two planes with respect to a point source in the object domain. Our calibration algorithm also determines the defocus distance between the planes.