High‐speed multiplane structured illumination microscopy of living cells using an image‐splitting prism

Super‐resolution structured illumination microscopy (SR‐SIM) can be conducted at video‐rate acquisition speeds when combined with high‐speed spatial light modulators and sCMOS cameras, rendering it particularly suitable for live‐cell imaging. If, however, three‐dimensional (3D) information is desired, the sequential acquisition of vertical image stacks employed by current setups significantly slows down the acquisition process. In this work, we present a multiplane approach to SR‐SIM that overcomes this slowdown via the simultaneous acquisition of multiple object planes, employing a recently introduced multiplane image splitting prism combined with high‐speed SIM illumination. This strategy requires only the introduction of a single optical element and the addition of a second camera to acquire a laterally highly resolved 3D image stack. We demonstrate the performance of multiplane SIM by applying this instrument to imaging the dynamics of mitochondria in living COS‐7 cells.