  The dimensions of neuronal synapses suggest that optical super-resolution
imaging methods are necessary for thorough investigation of protein
distributions and interactions. Nanoscopic evaluation of neuronal samples has
presented practical hurdles, but advancing methods are making synaptic protein
topology and quantification measurements feasible. This work explores the
application of Photoactivated Localization Microscopy (PALM) pointilistic
super-resolution imaging for investigation of the membrane bound sodium pump,
the Na,K-ATPase, in matured neurons.
  Two isoforms of the sodium pump (ATP1a1 and ATP1a3) were studied in cultured
neurons using the PALM-compatible fluorescent proteins PAGFP and mEos.
Nanoscopic imaging reveals a compartmentalized distribution of sodium pumps in
dendritic spines. Several nanoclusters of pumps are typically found in the
spine head and relatively few in the spine neck. The density of sodium pumps
was estimated from a quantification of detected single molecules to 600-800
pump copies/um2 in the spine heads.
  We have utilized PALM for dissection of nanoscale localization in mature
cultured neurons and demonstrated similar topology and quantification estimates
with PAGFP and mEos. PALM topology assessments of the sodium pump appeared
similar to previous STED studies, though quantification estimates varied
notably, implying that sample labeling strategies and choice of nanoscopic
imaging method are critical factors for correct quantification.
