PMID- 17416619
OWN - NLM
STAT- MEDLINE
DCOM- 20071016
LR  - 20240316
IS  - 0006-3495 (Print)
IS  - 1542-0086 (Electronic)
IS  - 0006-3495 (Linking)
VI  - 93
IP  - 3
DP  - 2007 Aug 1
TI  - Systematic colocalization errors between acridine orange and EGFP in astrocyte 
      vesicular organelles.
PG  - 969-80
AB  - 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.
FAU - Nadrigny, Fabien
AU  - Nadrigny F
AD  - INSERM, U603, Paris, France.
FAU - Li, Dongdong
AU  - Li D
FAU - Kemnitz, Klaus
AU  - Kemnitz K
FAU - Ropert, Nicole
AU  - Ropert N
FAU - Koulakoff, Annette
AU  - Koulakoff A
FAU - Rudolph, Stephanie
AU  - Rudolph S
FAU - Vitali, Marco
AU  - Vitali M
FAU - Giaume, Christian
AU  - Giaume C
FAU - Kirchhoff, Frank
AU  - Kirchhoff F
FAU - Oheim, Martin
AU  - Oheim M
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20070406
PL  - United States
TA  - Biophys J
JT  - Biophysical journal
JID - 0370626
RN  - 147336-22-9 (Green Fluorescent Proteins)
RN  - F30N4O6XVV (Acridine Orange)
SB  - IM
MH  - Acridine Orange/*analysis/*chemistry
MH  - Animals
MH  - Animals, Newborn
MH  - Astrocytes/*physiology/ultrastructure
MH  - Cerebral Cortex/physiology
MH  - Green Fluorescent Proteins/*chemistry
MH  - Kinetics
MH  - Mice
MH  - Mice, Inbred Strains
MH  - Microscopy, Fluorescence
MH  - Organelles/*physiology/ultrastructure
PMC - PMC1913145
EDAT- 2007/04/10 09:00
MHDA- 2007/10/17 09:00
PMCR- 2008/08/01
CRDT- 2007/04/10 09:00
PHST- 2007/04/10 09:00 [pubmed]
PHST- 2007/10/17 09:00 [medline]
PHST- 2007/04/10 09:00 [entrez]
PHST- 2008/08/01 00:00 [pmc-release]
AID - S0006-3495(07)71354-1 [pii]
AID - 102673 [pii]
AID - 10.1529/biophysj.106.102673 [doi]
PST - ppublish
SO  - Biophys J. 2007 Aug 1;93(3):969-80. doi: 10.1529/biophysj.106.102673. Epub 2007 
      Apr 6.