Cells are three-dimensional objects and intracellular motion is generally not constrained to a single plane. Multiplane microscopy allows visualization of cellular structures and dynamics through the simultaneous imaging of different focal planes within the sample. We realize multi-plane microscopy using image splitting prisms for ultrafast (ms), inter-plane drift-free 3D data acquisition. This implementation allows imaging across the visible spectrum with high trans-mission efficiency. Prism-based imaging is compatible with different microscopy modalities from conventional (multicolor) fluorescence, single-particle tracking and super-resolution mi-croscopy (SOFI, SIM) to label-free quantitative phase imaging. We are developing and apply-ing open-source multiplane imaging hardware and software to facilitate widespread adoption.

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