Systematic colocalization errors between acridine orange and EGFP in astrocyte vesicular organelles

Area of Science:

• Neuroscience
• Cell Biology
• Biophysics

Background:

• Dual-color imaging is used to study glutamatergic astrocytic secretory vesicles.
• Acridine orange (AO) and enhanced green fluorescent protein (EGFP) are common fluorophores in these studies.
• AO metachromasy can complicate the interpretation of dual-color fluorescence data.

Purpose of the Study:

• To investigate the reliability of distinguishing AO and EGFP fluorescence in dual-color imaging of astroglial vesicles.
• To determine if AO fluorescence interferes with EGFP detection.
• To propose an alternative imaging method for studying astroglial secretory vesicles.

Main Methods:

• Evanescent-field imaging with spectral fluorescence detection.
• Fluorescence lifetime imaging microscopy (FLIM).
• Spectral unmixing and calculation of fluorophore abundance maps.

Main Results:

• Green fluorescent AO monomers coexist with red fluorescing AO dimers in single astroglial vesicles.
• AO monomer emission spectrally overlaps with EGFP emission, causing false colocalization.
• Strong AO monomer fluorescence obscures EGFP signals, preventing EGFP detection.

Conclusions:

• Extreme caution is needed when quantifying colocalization of EGFP-tagged organelles with AO.
• AO's spectral properties interfere with the detection of EGFP in dual-color imaging.
• FM4-64/EGFP dual-color imaging is proposed as a reliable alternative for visualizing astroglial fusion-competent secretory vesicles.