This Article Full Text Full Text (PDF) All Versions of this Article: biophysj.106.102673v1 93/3/969    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nadrigny, F. Articles by Oheim, M. Search for Related Content PubMed PubMed Citation Articles by Nadrigny, F. Articles by Oheim, M.

Systematic Colocalization Errors between Acridine Orange and EGFP in Astrocyte Vesicular Organelles

Fabien Nadrigny ^*, Dongdong Li ^*, Klaus Kemnitz , Nicole Ropert ^*, Annette Koulakoff , Stephanie Rudolph , Marco Vitali , Christian Giaume , Frank Kirchhoff  and Martin Oheim ^*

^* INSERM, U603, Paris, France; Université Paris Descartes, Laboratory of Neurophysiology & New Microscopies, Paris, France; EuroPhoton, Berlin, Germany; INSERM, U640, Paris, France; Collège de France, Paris, France; and Neurogenetics, Max-Planck Institute of Experimental Medicine, Göttingen, Germany

Correspondence: Address reprint requests to Martin Oheim, Tel.: 33-1-42-86-42-21; E-mail: martin.oheim{at}univ-paris5.fr.

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.

This article has been cited by other articles:

				D. Li, N. Ropert, A. Koulakoff, C. Giaume, and M. Oheim Lysosomes Are the Major Vesicular Compartment Undergoing Ca2+-Regulated Exocytosis from Cortical Astrocytes J. Neurosci., July 23, 2008; 28(30): 7648 - 7658. [Abstract] [Full Text] [PDF]