Enhanced detection of fluorescence fluctuations for high-throughput super-resolution imaging

High-throughput super-resolution (SR) imaging is attractive for rapid and high-precision profiling in a wide range of biomedical applications. However, current SR methods require sophisticated acquisition optics and long integration times to acquire a single field of view. By exploiting the natural photophysics of fluorescence, fluctuation-based microscopy techniques can routinely break the diffraction limit without requiring additional optical components. However, their long acquisition time still poses a challenge for high-throughput imaging and the visualization of transient cellular dynamics. Here we propose super-resolution imaging based on autocorrelation with two-step deconvolution (SACD). Our method notably reduces the number of frames required by maximizing the detectable fluorescence fluctuation behaviour in each measurement. SACD requires only 20 frames to achieve a twofold improvement in lateral and axial resolution, whereas current SR optical fluctuation imaging (SOFI) needs more than 1,000 frames. With an acquisition time of ~10 min, we record SR images with 128-nm resolution over a field of view of 2.0 mm × 1.4 mm, which includes more than 2,000 cells. By applying the continuity and sparsity joint constraint, sparse deconvolution-assisted SACD enables four-dimensional imaging of live cells and events such as mitochondrial fission and fusion. Overall, as an open-sourced module, we anticipate that SACD will improve accessibility to SR imaging, thus facilitating biological studies of cells and organisms with high throughput and low cost. Super-resolution imaging based on autocorrelation with two-step deconvolution (SACD) enables recording super-resolution images with 128-nm spatial resolution over a field of view of 2.0 mm × 1.4 mm within a 10-min acquisition time.