Photoactivated Localization Microscopy (PALM) is one of the recently emerging techniques for optical imaging of protein distributions in biological samples at nanometer resolution. In PALM, numerous sparse subsets of photoactivatable fluorescent proteins are serially activated, localized, and bleached. The aggregate position information from all subsets is then assembled into a super-resolution image. In its initial realization, PALM used total internal reflection excitation (TIRF), thus the technique was limited to thin biological samples close to a microscope coverslip surface.
By combing PALM with temporal focusing, we have demonstrated that protein distributions deep (~10 μm) in cells with a lateral localization precision better than 50 nm could be imaged. Temporal focusing is a nonlinear optical technique whereby ultra-short laser pulses are focused in time. Using diffraction gratings and an appropriate geometry, it is possible to focus an optical pulse in the axial direction without focusing it in the lateral directions. Therefore, high spatial energy densities can be created in a widefield configuration, which can be used for two-photon excitation of fluorescent proteins. Using genetically expressed fluorescent proteins, this technique permits the study of protein-protein and other biological interactions at high resolutions in vivo. We have imaged amongst others proteins in the mitochondrial matrix and have resolved the cell membrane structure of Drosophila S2 cells in 3D. (Figure below).
Vaziri, A., Tang, J., Shroff, H., Shank, CV. (2008).
“Multilayer three-dimensional super- resolution imaging of thick biological samples.”
Proc Natl Acad Sci U S A. 105(51):20221-6 (Download)