Abstract
Single-molecule localization microscopy (SMLM) is a powerful super-resolution imaging technique that offers resolution far beyond the optical diffraction limit. The commonly used high numerical-aperture (NA) objective lenses in SMLM can only provide a nearly ideal point-spread function (PSF) at the center of the field-of-view (FOV), whereas the off-axis PSF is often distorted due to optical aberrations. Since precision and accuracy of three-dimensional (3D) spatial localization of single molecules heavily depend on the system's PSF, the FOV of 3D SMLM is often restricted to about 50 µm × 50 µm limiting its applications in visualizing intra-/intercellular interactions and high-throughput single-molecule analysis. Here we present a systematic study to show the influence of optical aberrations on large FOV 3D SMLM using unmodified, astigmatic, and double-helix PSFs. Our results show that optical aberrations introduce significant localization errors during image reconstruction and thereby produce unreliable imaging results at the corner of the FOV. To maximize SMLM's FOV, we proposed and verified the potential of using discrete field-dependent PSFs to retain precise and accurate single-molecule localization and compare their reconstruction results using simulated resolution test patterns and biological structures. Moreover, GPU acceleration empowers a discrete PSF calibration model with high localization speed, which can provide real-time SMLM image reconstruction. We envision these results will further guide the development of strategies that can provide real-time and reliable image reconstruction in large FOV 3D SMLM.