Temporal modulation and frame-differencing enhanced high-density super-resolution optical fluctuation imaging

Super-resolution microscopy has enabled nanoscale imaging beyond the diffraction limit. Among various techniques, super-resolution optical fluctuation imaging (SOFI) is notable for its hardware simplicity and suitability for live-cell imaging. However, under high fluorescent labeling density, SOFI reconstructions often suffer from reduced resolution and imaging artifacts. This study proposes a super-resolution optical fluctuation imaging approach based on temporal modulation and frame-differencing (dSOFI). By employing periodic photoactivation to modulate fluorophore density and introducing a frame-differencing algorithm, dSOFI effectively reduces molecular density during the reconstruction process. The method demonstrates broad compatibility across sample types, imaging modes, and post-processing algorithms. When integrated with the commercial spinning-disk confocal system, dSOFI achieves a lateral resolution of 72 nm in live-cell imaging, and reveals a potential upper bound of lateral resolution approaching ∼60 nm under well-controlled acquisition conditions when combined with sparse deconvolution. dSOFI provides a broadly applicable and easy-to-implement solution for high-density super-resolution imaging, particularly under live-cell conditions.