Abstract
Being able to use versatile light microscopy on live or fixed samples followed by electron microscopy imaging for high resolution analyses is a challenging goal. The advantage is of course that tracing and localizing fluorescently labeled molecules yields great information about dynamic cellular processes, while electron microscopy of the same sample provides exquisite information about subcellular structures. Here, I describe the straightforward combination of both methods by photoconversion of diaminobenzidine (DAB) through cyan fluorescent protein (CFP) tagged proteins localized to the Golgi apparatus in primary hippocampal neurons.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Lippincott-Schwartz J, Patterson GH (2009) Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging. Trends Cell Biol 19(11):555–565
Ward TH, Lippincott-Schwartz J (2006) The uses of green fluorescent protein in mammalian cells. Methods Biochem Anal 47:305–337
Giepmans BN, Adams S R, Ellisman MH, Tsien RY (2006) The fluorescent toolbox for assessing protein location and function. Science 312(5771):217–224
Grabenbauer M et al (2005) Correlative microscopy and electron tomography of GFP through photooxidation. Nat Methods 2(11): 857–862
Pellicciari C et al (2013) Ultrastructural detection of photosensitizing molecules by fluorescence photoconversion of diaminobenzidine. Histochem Cell Biol 139(6):863–871
Harata N, Ryan TA, Smith SJ, Buchanan J, Tsien RW (2001) Visualizing recycling synaptic vesicles in hippocampal neurons by FM 1-43 photoconversion. Proc Natl Acad Sci U S A 98(22):12748–12753
Opazo F, Rizzoli SO (2010) Studying synaptic vesicle pools using photoconversion of styryl dyes. J Vis Exp. 36
Dresbach T et al (2003) Functional regions of the presynaptic cytomatrix protein bassoon: significance for synaptic targeting and cytomatrix anchoring. Mol Cell Neurosci 23(2):279–291
Ohki EC, Tilkins ML, Ciccarone VC, Price PJ (2001) Improving the transfection efficiency of post-mitotic neurons. J Neurosci Methods 112(2):95–99
Fahimi HD, Baumgart E (1999) Current cytochemical techniques for the investigation of peroxisomes. A review. J Histochem Cytochem 47(10):1219–1232
Meiblitzer-Ruppitsch C et al (2008) Electron microscopic visualization of fluorescent signals in cellular compartments and organelles by means of DAB-photoconversion. Histochem Cell Biol 130(2):407–419
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Wittenmayer, N. (2014). Photoconversion of CFP to Study Neuronal Tissue with Electron Microscopy. In: Cambridge, S. (eds) Photoswitching Proteins. Methods in Molecular Biology, vol 1148. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0470-9_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-0470-9_6
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-0469-3
Online ISBN: 978-1-4939-0470-9
eBook Packages: Springer Protocols