Recently developed super-resolution optical imaging techniques, such as stochastic optical reconstruction microscopy (STORM) 3 and (fluorescent) photoactivated localization microscopy (FPALM/PALM) 4, 5, have redefined the resolution barrier and allowed cellular ultrastructures being resolved at ~7 nm resolution 6. Fluorescent imaging techniques have high specificity, but optical diffraction disqualifies conventional fluorescence microscopy for imaging the subcellular distribution and dynamics of high-density molecules. For instance, electron microscopy is unsuitable for dynamic imaging of a particular PPI subpopulation because of its poor specificity and low temporal resolution, albeit its exceeding spatial resolution. All of the current imaging approaches have limitations for studying PPIs in such small and crowded systems. For a given target protein, imaging its individual PPIs can be very challenging because of several inter-dependent issues, including multiple kinds of interacting partners, high molecule density and heterogonous dynamics, all imaged in a sub-diffraction cellular space.Ī typical example is the prokaryotic cell, which, although lacking internal membrane systems, is recently discovered to have subcellular domains and higher-order organization 2.
Meanwhile, accumulating evidence has shown that the functions of PPIs are tightly related to their spatial distribution and temporal dynamics, and therefore direct visualization of PPIs in living cells and organisms is crucial 2. Mass spectrum and biochemistry approaches have identified numerous PPI networks 1. Protein–protein interaction (PPI) is the foundation for most cellular processes.