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By switching fluorophores on and off in either a deterministic or astochastic manner, superresolution microscopy has enabled the imaging ofbiological structures at resolutions well beyond the diffraction limit.Superresolution optical fluctuation imaging (SOFI) provides an elegant way ofovercoming the diffraction limit in all three spatial dimensions by computinghigher-order cumulants of image sequences of blinking fluorophores acquiredwith a conventional widefield microscope. So far, three-dimensional (3D) SOFIhas only been demonstrated by sequential imaging of multiple depth positions.Here we introduce a versatile imaging scheme which allows for the simultaneousacquisition of multiple focal planes. Using 3D cross-cumulants, we show thatthe depth sampling can be increased. Consequently, the simultaneous acquisitionof multiple focal planes reduces the acquisition time and hence thephoto-bleaching of fluorescent markers. We demonstrate multiplane 3D SOFI byimaging the mitochondria network in fixed C2C12 cells over a total volume of$65\times65\times3.5 \mu\textrm{m}^3$ without depth scanning.

Multiplane 3D superresolution optical fluctuation imaging