Hajj, B. et al. Proc. Nat. Acad. Sci. USA 111, 17480–17485 (2014).

Super-resolution imaging techniques such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) are increasingly popular. Both techniques are typically carried out using total-internal-reflection fluorescence microscopes, however, which limit imaging depth. To achieve three-dimensional (3D) imaging, Hajj et al. implemented PALM/STORM using a multifocus microscope that can simultaneously acquire images from nine equally spaced focal planes. This approach allowed them to acquire images with high axial and lateral precision as well as to an imaging depth of 4 micrometers, which spans a typical mammalian cell and exceeds the depth reached by other super-resolution techniques. The researchers were able to generate super-resolution images of mitochondria in HeLa cells and two-color PALM/STORM images in yeast. This approach should open the door to many 3D super-resolution imaging experiments.