Dynamic live-cell super-resolution imaging with parallelized fluorescence emission difference microscopy

The investigation of the cell biology with fluorescence microscopy remains challenging because of the requirement of both high temporal and spatial resolution. In this paper, parallelized fluorescence emission difference microscopy (pFED) is presented to provide 2-fold increasing imaging speed than conventional FED and achieve high spatial resolution (0.2 lambda-0.3 lambda) with a novel system design. The super-resolution images are obtained by aligning and subtracting two images, that are acquired simultaneously using parallelized scanning solid- and donut-shaped excitation beams with a lateral offset between two foci. Herein, we demonstrate the general applicability of pFED with time-lapse imaging of various subcellular dynamics in live cells over an extended period.