Document Detail

Quantifying spatial organization in point-localization superresolution images using pair correlation analysis.
MedLine Citation:
PMID:  23348362     Owner:  NLM     Status:  MEDLINE    
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.
Prabuddha Sengupta; Tijana Jovanovic-Talisman; Jennifer Lippincott-Schwartz
Publication Detail:
Type:  Journal Article     Date:  2013-01-24
Journal Detail:
Title:  Nature protocols     Volume:  8     ISSN:  1750-2799     ISO Abbreviation:  Nat Protoc     Publication Date:  2013 Feb 
Date Detail:
Created Date:  2013-01-25     Completed Date:  2013-06-19     Revised Date:  2014-02-20    
Medline Journal Info:
Nlm Unique ID:  101284307     Medline TA:  Nat Protoc     Country:  England    
Other Details:
Languages:  eng     Pagination:  345-54     Citation Subset:  IM    
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MeSH Terms
Green Fluorescent Proteins
Image Processing, Computer-Assisted*
Intracellular Space / metabolism*
Membrane Proteins / metabolism*
Microscopy / methods*
Grant Support
Reg. No./Substance:
0/Membrane Proteins; 147336-22-9/Green Fluorescent Proteins

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