Quantifying spatial organization in point-localization superresolution images using pair correlation analysis.
The distinctive distributions of proteins within subcellular compartments both at steady state and during signaling events have essential roles in cell function. Here we describe a method for delineating the complex arrangement of proteins within subcellular structures visualized using point-localization superresolution (PL-SR) imaging. The approach, called pair correlation photoactivated localization microscopy (PC-PALM), uses a pair-correlation algorithm to precisely identify single molecules in PL-SR imaging data sets, and it is used to decipher quantitative features of protein organization within subcellular compartments, including the existence of protein clusters and the size, density and number of proteins in these clusters. We provide a step-by-step protocol for PC-PALM, illustrating its analysis capability for four plasma membrane proteins tagged with photoactivatable GFP (PAGFP). The experimental steps for PC-PALM can be carried out in 3 d and the analysis can be done in ∼6-8 h. Researchers need to have substantial experience in single-molecule imaging and statistical analysis to conduct the experiments and carry out this analysis.
The Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA.
Prabuddha
P
Sengupta
Tijana
T
Jovanovic-Talisman
Jennifer
J
Lippincott-Schwartz
D000465:Algorithms; D049452:Green Fluorescent Proteins; D007091:Image Processing, Computer-Assisted; D042541:Intracellular Space / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008853:Microscopy / Q000379:methods*
D008565:Membrane Proteins; D049452:Green Fluorescent Proteins