Document Detail

Systematic colocalization errors between acridine orange and EGFP in astrocyte vesicular organelles.
MedLine Citation:
PMID:  17416619     Owner:  NLM     Status:  MEDLINE    
Dual-color imaging of acridine orange (AO) and EGFP fused to a vesicular glutamate transporter or the vesicle-associated membrane proteins 2 or 3 has been used to visualize a supposedly well-defined subpopulation of glutamatergic astrocytic secretory vesicles undergoing regulated exocytosis. However, AO metachromasy results in the concomitant emission of green and red fluorescence from AO-stained tissue. Therefore, the question arises whether AO and EGFP fluorescence can be distinguished reliably. We used evanescent-field imaging with spectral fluorescence detection as well as fluorescence lifetime imaging microscopy to demonstrate that green fluorescent AO monomers inevitably coexist with red fluorescing AO dimers, at the level of single astroglial vesicles. The green monomer emission spectrally overlaps with that of EGFP and produces a false apparent colocalization on dual-color images. On fluorophore abundance maps calculated from spectrally resolved and unmixed single-vesicle spectral image stacks, EGFP is obscured by the strong green monomer fluorescence, precluding the detection of EGFP. Hence, extreme caution is required when deriving quantitative colocalization information from images of dim fluorescing EGFP-tagged organelles colabeled with bright and broadly emitting dyes like AO. We finally introduce FM4-64/EGFP dual-color imaging as a remedy for imaging a distinct population of astroglial fusion-competent secretory vesicles.
Fabien Nadrigny; Dongdong Li; Klaus Kemnitz; Nicole Ropert; Annette Koulakoff; Stephanie Rudolph; Marco Vitali; Christian Giaume; Frank Kirchhoff; Martin Oheim
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2007-04-06
Journal Detail:
Title:  Biophysical journal     Volume:  93     ISSN:  0006-3495     ISO Abbreviation:  Biophys. J.     Publication Date:  2007 Aug 
Date Detail:
Created Date:  2007-07-09     Completed Date:  2007-10-16     Revised Date:  2013-06-06    
Medline Journal Info:
Nlm Unique ID:  0370626     Medline TA:  Biophys J     Country:  United States    
Other Details:
Languages:  eng     Pagination:  969-80     Citation Subset:  IM    
INSERM, U603, Paris, France.
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MeSH Terms
Acridine Orange / analysis*,  chemistry*
Animals, Newborn
Astrocytes / physiology*,  ultrastructure
Cerebral Cortex / physiology
Green Fluorescent Proteins / chemistry*
Mice, Inbred Strains
Microscopy, Fluorescence
Organelles / physiology*,  ultrastructure
Reg. No./Substance:
147336-22-9/Green Fluorescent Proteins; 65-61-2/Acridine Orange

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