Light microscopy has given biologists eyes that can peer into small structures in cells and tissues. But owing to diffraction, a wave of light cannot be focused to an arbitrarily small point. Conventional light microscopes therefore have been long thought to be uncapable of resolving two objects closer together than about half the wavelength of light, with an imaging resolution of approximately 200 nanometers laterally and 600 nanometers axially.

A 3D STORM image of the mitochondrial network in a cell (Nat. Methods 5, 1047–1052; 2008).

Over the last several years, however, super-resolution techniques that allow the acquisition of microscopy images with lateral resolution on the order of tens of nanometers have emerged. Super-resolution fluorescence microscopy has enabled unprecedented resolution for several applications, for instance, live-cell imaging of dynamic cellular structures such as dendritic spines; indeed, it was the Nature Methods' choice for Method of the Year 2008.

But biological specimens such as cells are inherently three-dimensional (3D) objects, and early super-resolution methods were limited to improving the resolution either axially or laterally but not along all three axes simultaneously. Not only is it desirable to obtain a super-resolved image along all three axes, but imaging in three dimensions is critical for obtaining dynamic information in living cells. For both 3D and fast,live-cell imaging, current super-resolution techniques often fall short.

In recent years, the axial resolution of super-resolution techniques such as those based on stimulated emission depletion microscopy (STED), stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM) has considerably improved, and in some cases it is now possible to attain sub-20-nanometer resolution in all three dimensions. This additional information comes at the cost of longer imaging times though. Slow acquisition speeds already limit live-cell, two-dimensional super-resolution imaging applications, and improvements are needed so that thick samples with dimensions closer to the full thickness of a cell can be imaged at nanoscale resolution along all three axes and at speeds sufficient to apply these methods to living whole cells.

The eventual arrival of high-speed, 3D super-resolution fluorescence imaging will allow these techniques to be increasingly applied to image the dynamic cellular environment at unprecedented levels of detail, fulfilling their potential as tools of unquestionable value to cell biologists.