Not yet reviewed by Pith; the record is open.

This study introduces a structural-based training approach for CNN-based algorithms in single-molecule localization microscopy (SMLM) and 3D object reconstruction. We compare this approach with the traditional random-based training method, utilizing the LUENN package as our AI pipeline. The quantitative evaluation demonstrates significant improvements in detection rate and localization precision with the structural-based training approach, particularly in varying signal-to-noise ratios (SNRs). Moreover, the method effectively removes checkerboard artifacts, ensuring more accurate 3D reconstructions. Our findings highlight the potential of the structural-based training approach to advance super-resolution microscopy and deepen our understanding of complex biological systems at the nanoscale.