Monitoring rapid endocytosis in the electron microscope via photoconversion of vesicles fluorescently labeled with FM1-43.
Cells communicate via endo- and exocytosis with their environment and neighboring cells. At synapses of the nervous system, fast exocytosis is coupled to fast endocytosis, which forms the basis for neurotransmitter release. The introduction of the unique fluorescent FM dyes allowed the monitoring of fast synaptic vesicle exo-endocytic cycling during live imaging sessions and after photoconversion of FM dyes into an electron-dense diaminobenzidine polymer synaptic vesicle cycling can be studied in the electron microscope. This protocol describes FM dye labeling of synaptic vesicles of cultured hippocampal neurons and photoconversion of the fluorescent synaptic vesicles for analysis in the electron microscope (EM).
Schikorski
Thomas
T
Neuroscience Department, Universidad Central del Caribe, Bayamon, Puerto Rico.
D000818:Animals; D001253:Astrocytes / Q000737:chemistry / Q000166:cytology; D002478:Cells, Cultured; D004705:Endocytosis; D005456:Fluorescent Dyes / Q000737:chemistry*; D006624:Hippocampus / Q000166:cytology; D008854:Microscopy, Electron; D009474:Neurons / Q000737:chemistry / Q000166:cytology; D055668:Photochemical Processes; D011726:Pyridinium Compounds / Q000737:chemistry*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*; D051381:Rats; D013194:Staining and Labeling; D013997:Time Factors
C073804:FM1 43; D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds