By switching fluorophores on and off in either a deterministic or a
stochastic manner, superresolution microscopy has enabled the imaging of
biological structures at resolutions well beyond the diffraction limit.
Superresolution optical fluctuation imaging (SOFI) provides an elegant way of
overcoming the diffraction limit in all three spatial dimensions by computing
higher-order cumulants of image sequences of blinking fluorophores acquired
with a conventional widefield microscope. So far, three-dimensional (3D) SOFI
has only been demonstrated by sequential imaging of multiple depth positions.
Here we introduce a versatile imaging scheme which allows for the simultaneous
acquisition of multiple focal planes. Using 3D cross-cumulants, we show that
the depth sampling can be increased. Consequently, the simultaneous acquisition
of multiple focal planes reduces the acquisition time and hence the
photo-bleaching of fluorescent markers. We demonstrate multiplane 3D SOFI by
imaging the mitochondria network in fixed C2C12 cells over a total volume of
65×65×3.5μm3 without depth scanning.Comment: 7 pages, 3 figure