Single-molecule localization microscopy (SMLM) in a typical wide-field setup has been widely used for investigating sub-cellular structures with super resolution. However, field-dependent aberrations restrict the field of view (FOV) to only few tens of micrometers. Here, we present a deep learning method for precise localization of spatially variant point emitters (FD-DeepLoc) over a large FOV covering the full chip of a modern sCMOS camera. Using a graphic processing unit (GPU) based vectorial PSF fitter, we can fast and accurately model the spatially variant point spread function (PSF) of a high numerical aperture (NA) objective in the entire FOV. Combined with deformable mirror based optimal PSF engineering, we demonstrate high-accuracy 3D SMLM over a volume of ~180 × 180 × 5 μm 3 , allowing us to image mitochondria and nuclear pore complex in the entire cells in a single imaging cycle without hardware scanning -a 100-fold increase in throughput compared to the state-of-the-art.

Point spread function (PSF) engineering is an important technique to encode the properties (e.g., 3D positions, color, and orientation) of a single molecule in the shape of the PSF, often with the help of a programmable phase modulator. A deformable mirror (DM) is currently the most widely used phase modulator for fluorescence detection as it shows negligible photon loss. However, it relies on careful calibration for precise wavefront control. Therefore, design of an optimal PSF not only relies on the theoretical calculation of the maximum information content, but also the physical behavior of the phase modulator, which is often ignored during the optimization process. Here, we develop a framework for PSF engineering which could generate a device specific optimal PSF for 3D super-resolution imaging using a DM. We use our method to generate two types of PSFs with depths of field comparable to the widely used astigmatism and tetrapod PSFs, respectively. We demonstrate the superior performance of the DM specific optimal PSF over the conventional astigmatism and tetrapod PSF both theoretically and experimentally.

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