Pixel-reassigned line-scanning microscopy for fast volumetric super-resolution imaging.

Super-resolution microscopy has revolutionized the field of biophotonics by revealing detailed 3D biological structures. Nonetheless, the technique is still largely limited by the low throughput and hampered by increased background signals for dense or thick biological specimens. In this paper, we present a pixel-reassigned continuous line-scanning microscope for large-scale high-speed 3D super-resolution imaging, which achieves an imaging resolution of 0.41 µm in the lateral direction, i.e., a 2× resolution enhancement from the raw images. Specifically, the recorded line images are first reassigned to the line-excitation center at each scanning position to enhance the resolution. Next, a modified HiLo algorithm is applied to reduce the background signals. Parametric models have been developed to simulate the imaging results of randomly distributed fluorescent beads. Imaging experiments were designed and performed to verify the predicted performance on various biological samples, which demonstrated an imaging speed of 3400 pixels/ms on millimeter-scale specimens. These results confirm the pixel-reassigned line-scanning microscopy is a facile and powerful method to realize large-area super-resolution imaging on thick or dense biological samples.