Simultaneous Multicolor Multifocal Scanning Microscopy

[Image: see text] Super-resolution fluorescence microscopy has revolutionized cell biology over the past decade, enabling the visualization of subcellular complexity with unparalleled clarity and detail. However, the rapid development of image-scanning-based super-resolution systems still restrains convenient access to commonly used instruments such as epi-fluorescence microscopes. Here, we present multifocal scanning microscopy (MSM) for super-resolution imaging with simultaneous multicolor acquisition and minimal instrumental complexity. MSM implements a stationary, interposed multifocal multicolor excitation by exploiting the motion of the specimens, realizing super-resolution microscopy through a general epi-fluorescence platform without compromising the image-scanning mechanism or inducing complex instrument alignment. The system is demonstrated with various phantom and biological specimens, and the results present effective resolution doubling, optical sectioning, and contrast enhancement. We anticipate MSM, as a highly accessible and compatible super-resolution technique, to offer a promising methodological pathway for broad cell biological discoveries.