Vaziri et al describe a process by which they improved upon photoactivated localization microscopy to enable depth imaging with a resolution greater than 50 nm. The team used temporal focusing, whereby they targeted two spatially overlapping two-photon beams, one at the photoactive flourescent proteins and one at the two-photon excitation peak. This enabled them to eliminate noise from background layers.
In imaging there is always a trade-off between the number of molecules imaged and the resolution. Including more molecules leads to a lower resolution, while including less molecules (only the bright ones) leads to a higher resolution. In order to enable a higher resolution, they were only able to sample around 80% of the fruit fly’s Dronpa molecules. Their novel technique will surely be used in studies of the neural circuitry of Drospholia in the near future.
Vaziri A, Tang J, Shroff H, Shank CV. 2008 Multilayer three-dimensional super resolution imaging of thick biological samples. PNAS 105:20221-20226.