%0 Journal Article %1 Iliev2007a %A Iliev, A. I. %A Wouters, F. S. %D 2007 %J J Neurosci Methods %K *Microscopy, *Photobleaching After Animals Axons/*physiology Biological C57BL Confocal Cultured Embryo, Fluorescence Hippocampus/cytology Luminescent Mammalian Mice Neurons/*ultrastructure Photobleaching/*methods Proteins/metabolism Recovery Transport Cell %N 1 %P 39-46 %T Application of simple photobleaching microscopy techniques for the determination of the balance between anterograde and retrograde axonal transport %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17123628 %V 161 %X 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.

@article{Iliev2007a, abstract = {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.}, added-at = {2010-12-14T18:12:02.000+0100}, author = {Iliev, A. I. and Wouters, F. S.}, biburl = {https://www.bibsonomy.org/bibtex/2e5726b2075ca4094335a2c32192231b1/pharmawuerz}, endnotereftype = {Journal Article}, interhash = {e66c326da05efa0cc9f27472825452b3}, intrahash = {e5726b2075ca4094335a2c32192231b1}, issn = {0165-0270 (Print) 0165-0270 (Linking)}, journal = {J Neurosci Methods}, keywords = {*Microscopy, *Photobleaching After Animals Axons/*physiology Biological C57BL Confocal Cultured Embryo, Fluorescence Hippocampus/cytology Luminescent Mammalian Mice Neurons/*ultrastructure Photobleaching/*methods Proteins/metabolism Recovery Transport Cell}, month = {Mar 30}, note = {Iliev, Asparouh I Wouters, Fred S Research Support, Non-U.S. Gov't Netherlands Journal of neuroscience methods J Neurosci Methods. 2007 Mar 30;161(1):39-46. Epub 2006 Nov 22.}, number = 1, pages = {39-46}, shorttitle = {Application of simple photobleaching microscopy techniques for the determination of the balance between anterograde and retrograde axonal transport}, timestamp = {2010-12-14T18:22:13.000+0100}, title = {Application of simple photobleaching microscopy techniques for the determination of the balance between anterograde and retrograde axonal transport}, url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17123628}, volume = 161, year = 2007 }