Parallel image-scanning autocorrelation-deconvolution microscopy

We propose parallel image-scanning autocorrelation-deconvolution microscopy (PISADM), a multifocal-scanning strategy that converts global reconstruction into spatially localized reconstructions. By combining spatially localized multifocal excitation with scanning-position-wise SACD reconstruction and focus-wise pixel reassignment, PISADM reduces the effective accumulation of finite-frame cross terms and improves reconstruction stability in dense regions, thereby enhancing the separability of closely spaced structures. Monte-Carlo simulations show that, at an SBR of 10 dB, PISADM reduces the stable separability limit to approximately 100-110 nm, compared with about 140-150 nm for SACD under the same conditions. Fourier ring correlation analysis shows that PISADM preserves more reliable high-frequency information near the separability limit. In two-dimensional, long-term, and three-dimensional imaging of PCSTAR-labeled microtubules, PISADM consistently provides clearer separation of neighboring filaments while better preserving structural fidelity. These results indicate that PISADM provides a practical strategy for resolving densely packed structures with small spatial separations in super-resolution imaging.