ArticleBiophysical journal2007
Systematic colocalization errors between acridine orange and EGFP in astrocyte vesicular organelles.
Article in Biophysical journal, 2007. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 15 papers.
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Who cites it
15 citing papers in PubMed, 37 citations in OpenAlex.
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- A Light-Sheet-Based Imaaging Spectrometer to Characterize Acridine Orange Fluorescence within Leukocytes.Diagnostics (Basel, Switzerland) · 2020Article
- Fish telocytes and their relation to rodlet cells in ruby-red-fin shark (rainbow shark) Epalzeorhynchos frenatum (Teleostei: Cyprinidae).Scientific reports · 2020Article
- Morphological and immunohistochemical phenotype of TCs in the intestinal bulb of Grass carp and their potential role in intestinal immunity.Scientific reports · 2020Article
- Morphological changes of telocytes in camel efferent ductules in response to seasonal variations during the reproductive cycle.Scientific reports · 2019Article
- Astrocyte VAMP3 vesicles undergo Ca2+ -independent cycling and modulate glutamate transporter trafficking.The Journal of physiology · 2015Article
- New tools for investigating astrocyte-to-neuron communication.Frontiers in cellular neuroscience · 2013Review
- Lack of evidence for vesicular glutamate transporter expression in mouse astrocytes.The Journal of neuroscience : the official journal of the Society for Neuroscience · 2013Article
- Optogenetic activation of LiGluR-expressing astrocytes evokes anion channel-mediated glutamate release.The Journal of physiology · 2012Article
- Fast subplasma membrane Ca2+ transients control exo-endocytosis of synaptic-like microvesicles in astrocytes.The Journal of neuroscience : the official journal of the Society for Neuroscience · 2008Article
- Lysosomes are the major vesicular compartment undergoing Ca2+-regulated exocytosis from cortical astrocytes.The Journal of neuroscience : the official journal of the Society for Neuroscience · 2008Article
- Resolving vesicle fusion from lysis to monitor calcium-triggered lysosomal exocytosis in astrocytes.Proceedings of the National Academy of Sciences of the United States of America · 2007Article
Corrections and comments
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Authors and funding
10 authors at 6 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.