One of the ultimate goals of superresolution microscopy is to investigate the dynamics of interacting proteins in living cells at spatial resolutions that far exceed those of traditional diffraction-limited techniques. Most of the studies conducted by STED, GSD, PALM, STORM, and related techniques have utilized fixed cells and tissues where dynamic studies are impossible. Emerging methodologies for observing living cells at high resolution are just beginning to surface.
Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories. Proceedings of the National Academy of Sciences (USA) 104: 17370-17375 (2007). A report from the inventors of FPALM, Dr. Hess and colleagues investigate the dynamics of hemagglutinin distribution in membranes at 40-nanometer resolution in living and fixed fibroblast cells.
Manley, S., Gillette, J. M., Patterson, G. H., Shroff, H., Hess, H. F., Betzig, E. and Lippincott-Schwartz, J.
High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nature Methods 5: 155-168 (2008). The authors combine PALM with live-cell single-particle tracking to create a new method termed sptPALM. Spatially resolved maps of single-molecule motions in membrane proteins were investigated to determine trajectories.
Video-rate far-field optical nanoscopy dissects synaptic vesicle movement. Science 320: 246-249 (2008). In one of the most elegant live-cell imaging demonstrations yet reported, Dr. Hell and co-workers gather near video-rate imaging of vesicle mobility in synaptic boutons at 62-nanometer resolution.
Structured illumination microscopy of a living cell. European Biophysics Journal 38: 807-812 (2009). The authors investigate live-cell imaging with structured illumination on living COS-1 (African Green monkey kidney) cells labeled with MitoTracker Red. Time-lapse images were gathered at three-minute intervals to produce videos of the clustered organelles.
Differential evanescence nanometry: Live-cell fluorescence measurements with 10-nm axial resolution on the plasma membrane. Biophysical Journal 94: 2333-2342 (2008). A resolution of 10 nanometers was achieved by simultaneous tracking the average axial positions of diffraction-limited objects with two different sets of fluorophores. The method was used to follow the location of clathrin and its adaptor, AP-2.
Super-resolution video microscopy of live cells by structured illumination. Nature Methods 6: 339-354 (2009). A high-speed structured illumination instrument capable of 100-nanometer resolution at frame rates up to 11 Hertz is described. The microscope was used to demonstrate imaging of tubulin and kinesin dynamics in living insect cells.
Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics. Nature Methods 5: 417-423 (2008). Eric Betzig and co-workers apply PALM imaging to living cells expressing a fusion of tandem dimer Eos fluorescent protein to paxillin. They achieved a spatial resolution of approximately 60 nanometers and a frame rate of 25 seconds.
Investigating intracellular dynamics of FtsZ cytoskeleton with photoactivation single-molecule tracking. Biophysical Journal 95: 2009-2016 (2008). Using living E. coli cells, the authors demonstrate photoactivation single-molecule tracking of mobility dynamics of intracellular bacterial cytoskeleton FtsZ protein molecules.
Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP. Nature Methods 5: 947-956 (2008). By combining photo-induced activation of single EYFP molecules and time-lapse imaging, the authors obtained sub-40 nanometer resolution images of the filamentous superstructure of the bacterial protein MreB in living cells.
Single-molecule and superresolution imaging in live bacteria cells. Cold Spring Harbor Perspectives in Biology 2: a000448 (2010). An excellent review article on the progress of live-cell imaging using superresolution techniques in live bacteria, one of the most difficult specimens. The authors discuss instrument methodology and theory, data processing, and diffusion dynamics.