Three-dimensional super-resolution imaging

There are fewer topics hotter in optical microscopy than breaking the diffraction limit. Scientists around the world have been making a push in the last few years to lessen the drawbacks to various super-resolution techniques. Today, Jian Yong Tang from Howard Hughes Medical Institute presented a way to add the third dimension to super-resolution imaging.

His method, which he calls virtual volume photoactivated localization microscopy (PALM), bridges the gap in current methods, most of which provide either a high volume or high axial resolution but not both. He used a simple approach – placing a tilted mirror beside the sample – to create a virtual image of the side of the sample. The microfabricated tilted micro-mirror simultaneously generates a side view of emitting single molecules. This converts the task of axial localization to lateral localization. Thus, the 3D image can be reconstructed with isotropic resolution by correlating single molecule events in the front view and side views.

He tried this method on bacteria, imaging a protein labeled with Dronpa (a photoswitchable fluorescent protein) in a single E. coli cell. He was able to produce very nice 3D images that were higher resolution than confocal microscopy would produce.

Virtual volume PALM comes with the advantages of not relying on an off-focus pattern, being less sensitive to Z-drift, being less vulnerable to optical aberration, and producing similar resolutions in all dimensions. It does have a limited field of view, but this can be overcome using tiling. For biological applications, the third dimension helps give assurance that the image or shape being seen isn’t an artifact.

Tang also presented results from combining virtual volume PALM with another technique called point accumulation for imaging in nanoscale topography (PAINT) and would like to combine it with other methods such as temporal focusing. In addition, his group is working gradually toward live cell imaging.