Superresolution techniques such as photoactivated localization microscopy (PALM) enable the imaging of fluorescent protein chimeras to reveal the organization of genetically-expressed proteins on the nanoscale with a density of molecules high enough to provide structural context. In PALM, serial photoactivation and subsequent bleaching of numerous sparse subsets of photoactivated fluorescent protein molecules is performed. Individual molecules are then localized at near molecular resolution by determining their centers of fluorescent emission via a statistical fit of their point-spread-function. The aggregate position information from all subsets is then assembled into a super-resolution image, in which individual fluorescent molecules are isolated at high molecular densities (up to 10,000 molecules/micron squared). We have previously demonstrated PALM imaging of intracellular structures (including lysosome, Golgi apparatus and mitochondria) in cryo-prepared thin sections, as well as imaging of vinculin and actin in fixed cells with TIRF excitation, and correlative PALM/transmission electron microscopy of a mitochondrial marker protein. ? ? We have developed a dual-label PALM assay system using two different photactivatable molecules expressed within cells. In addition, we have developed a system for doing single particle tracking using PALM in living cells that allows protein diffusion and immobilization to be characterized at the single molecule level. Called single particle tracking PALM (sptPALM), the technique involves activating, localizing and bleaching many subsets of photoactivatated fluorescent protein chimeras in live cells. Spatially-resolved maps of single molecule motions can be obtained by imaging membrane proteins with this technique, providing several orders of magnitude more trajectories per cell than by traditional single particle tracking. By probing distinct subsets of molecules, including Gag and VSVG, we demonstrated that sptPALM can provide a powerful means for exploring the origin of spatial and temporal heterogeneities in membranes.? ? Another fluorescent protein technique developed in our lab allows protein topology to be determined in living cells. Termed fluorescence protease protection (FPP), the assay provides a fluorescent readout in response to trypsin-induced destruction of GFP attached to a protein-of-interest before and after plasma membrane permeabilization. In performing the FPP assay, a fluorescent protein is attached to the N- or C-terminus of a protein of interest. Subsequently, cells expressing the fusion protein are exposed to trypsin either before or after plasma membrane permeabilization by digitonin. If the fluourescent protein moiety on the expressed protein faces the environment exposed to trypsin (that is the cytoplasm), then its fluorescent signal will be lost. Conversely, if the fluorescent protein moiety on the expressed protein faces the environment protected from trypsin (that is, the lumen of a compartment) then its fluorescence persists. Given these outcomes and the fluorescent proteins known engineered position within the protein, it is possible to deduce the orientation of the protein across the lipid bilayer. We demonstrated the broad applicability of FPP by using it to define the topology of proteins localized to several different organelles, including the ER, Golgi apparatus, mitochondria, peroxisomes and autophagosomes.
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