Dynamic (Live Cell) FPALM

One excellent advantage of FPALM is that it allows the super-resolution imaging of proteins in living cells. By keeping samples on a staged heated to 37 degrees Celsius, with 5% carbon dioxide, we can keep cells in physiological conditions while we image. This allows the investigation of dynamic processes, including the mapping of the paths taken (trajectories) of individual molecules as they move throughout the cell.


FPALM was the first localization microscopy technique to be applied to live cells (Hess et al 2007). In live cells, in addition to providing a time dependent spatial map of the locations of labeled molecules, FPALM can track single molecules over multiple frames, and their trajectories determined over timescales of milliseconds. Thus, FPALM provides access to short timescales and nanoscale resolution.

Live cell Figure 2 AI CS October 9 2012 Gudheti et al 17cm wide
Living fibroblast cell (A) imaged by FPALM (boxed region shown in B). Dendra2-hemagglutinin was imaged for tens of seconds (C). In (D), PAmCherry-actin (red) was imaged with Dendra2-HA (white); the mobility of HA (white arrows) is associated with the surrounding actin density (see graphs in E and F).




Recent Dynamic FPALM Publications

Gudheti M.*, Curthoys N.*, Gould T.*, Kim D.*, Gabor K.*, Gunewardene M.*, Zimmerberg J., Gosse J., Kim C., Hess S.T.*, “Actin Mediates the Nanoscale Membrane Organization of the Clustered Membrane Protein Influenza Hemagglutinin,” Biophysical Journal 104:2182-92, 2013. PMCID: 3660645 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3660645/

Hess, S.T.*, Gould, T.J.*, Gudheti, M.V.*, Maas, S.A., Mills, K.D., and Zimmerberg, J. “Dynamic Clustered Distribution of Hemagglutinin Resolved at 40 nm in Living Cell Membranes Discriminates Between Raft Theories,” PNAS 104:17370-5, 2007.

* denotes former and current members of the Hess Lab