Resolution-enhanced multifocal structured illumination microscopy using fluorescence fluctuations and Fourier ptychography scheme.

Abstract

Multifocal structured illumination microscopy (MSIM) provides a twofold resolution enhancement over the optical diffraction limit at depths of up to 50 μm in samples. This is achieved through sparse multifocal excitation patterns and digital image post-processing, making MSIM a highly advantageous technique for the three-dimensional super-resolution (SR) imaging of thick specimens. However, the spatial resolution of MSIM is inherently constrained by its underlying imaging principles. This paper presents what we believe to be a novel method that integrates SR optical fluctuation imaging based on Fourier ptychography and deconvolution (SFPD) with MSIM, termed SFPD-MSIM. Using photoblinking InP/ZnSe/ZnS core-shell quantum dot fluorescent probes for sample labeling, we demonstrate that, compared to wide-field imaging microscopy, SFPD-MSIM achieves fourfold resolution improvement. Additionally, it substantially reduces the image-acquisition time while preserving the structural integrity of the original samples. This advancement marks a major step forward in MSIM technology, providing a powerful tool for detailed structural analysis of complex and thick biological specimens.