Application of simple photobleaching microscopy techniques for the determination of the balance between anterograde and retrograde axonal transport.
The directionality of axonal transport represents an important question in neurophysiological and neuropathological research. Various approaches such as videomicroscopy, radioisotopic and fluorescence-based techniques are used. Recently, a novel FRAP-based (fluorescent recovery after photobleaching) technique using synaptophysin-EGFP expression in primary neurons was applied, allowing reliable and sensitive evaluation of gross axonal transport changes using confocal live-imaging microscopy. Here, we describe a novel FLIP-based (fluorescence loss in photobleaching) approach using a synaptophysin-EGFP probe that allows the differential evaluation of the ante- and retrograde transport parameters. Furthermore, we improved the sensitivity of the probe by substituting EGFP with an ECFP/VenusYFP fusion FRET (fluorescence resonance energy transfer) pair. The use of this FRET couple improves the precision of axonal transport measurements by combining FLIP and FLAP (fluorescence localization after photobleaching) techniques and eliminating the need for pre-bleaching images and thus pixel shifts between various exposures, and by reducing the deleterious effect of photobleaching.
Iliev
Asparouh I
AI
Cell Biophysics Group, European Neuroscience Institute-Goettingen, Medical Faculty, Georg August University-Goettingen, Waldweg 33, 37073 Goettingen, Germany. ailiev@gwdg.de
Wouters
Fred S
FS
D000818:Animals; D001369:Axons / Q000502:physiology*; D001692:Biological Transport; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D006624:Hippocampus / Q000166:cytology; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D009474:Neurons / Q000648:ultrastructure*; D038761:Photobleaching
D008164:Luminescent Proteins