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enforcement a consistent PSF response through large depths, therefore permitting robust 3D-super-resolution volumetric imaging of fluorescence stained whole-cell samples or tissues. We show that our method allows imaging through 30 mm brain sections from the mouse frontal cortex by reconstructing fibrillar amyloid-b plaques found in Alzheimer's disease. . Currently, it is highly desirable but still challenging to obtain high-resolution (<50 nm) three-dimensional (3D) super-resolution information of structures in fixed specimens as well as for dynamic processes in live cells. Here we introduce a simple approach, without using 3D super-resolution microscopy or realtime 3D particle tracking, to estimate 3D sub-diffraction-limited structural or dynamic information in rotationally symmetric bio-structures. This is a postlocalization analysis that transforms 2D super-resolution images or 2D single-molecule localization distributions into their corresponding 3D spatial probability distributions based on prior known structural knowledge. This analysis is ideal in cases where the ultrastructure of a cellular structure is known but the sub-structural localization of a particular (usually mobile) protein is not. The method has been successfully applied to achieve 3D structural and functional sub-diffraction-limited information for 25-300 nm subcellular organelles that meet the rotational symmetry requirement, such as nuclear pore complex, primary cilium and microtubule. Herein, we will provide comprehensive analyses of this method by using experimental data and computational simulations.

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