Abstract
With the live imaging of embryos, the dynamics of developmental processes, such as tissue remodeling, cell morphogenesis, and, campus protein dynamics can be observed and quantified. This has greatly improved the mechanistic understanding of biological processes. Here we describe how embryos can be prepared for imaging mainly, but not only, fluorescent proteins and probes. This chapter is a users’ guide that addresses the following aspects of fluorescent embryo imaging: (1) How to handle and prepare embryos for live microscopy. (2) What microscopic setups are available for embryo imaging and what should they be used for. (3) How to practically use fluorescent imaging setups depending on the experimental context: large-scale imaging of multiple embryos, high-resolution four-dimensional imaging of single embryos, studies of protein dynamics, and so on. (4) Finally, we focus on pitfalls and how to overcome a variety of possible problems encountered during live imaging.
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 subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Brand, A. H. and Perrimon, N. (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415.
Morin, X., Daneman, R., Zavortink, M., and Chia, W. (2001) A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila. Proc. Natl. Acad. Sci. USA 98, 15,050–15,055.
Kelso, R. J., Buszczak, M., Quinones, A. T., Castiblanco, C., Mazzalupo, S., and Cooley, L. (2004) Flytrap, a database documenting a GFP protein-trap insertion screen in Drosophila melanogaster. Nucleic Acids Res. 32, D418–D420.
Pilot, F., Philippe, J. M., Lemmers, C., Chauvin, J. P., and Lecuit, T. (2006) Developmental control of nuclear morphogenesis and anchoring by charleston, identified in a functional genomic screen of Drosophila cellularisation. Development 133, 711–723.
Bertet, C., Sulak, L., and Lecuit, T. (2004) Myosin-dependent junction remodelling controls planar cell intercalation and axis elongation. Nature 429, 667–671.
Sano, H., Renault, A. D., and Lehmann, R. (2005) Control of lateral migration and germ cell elimination by the Drosophila melanogaster lipid phosphate phosphatases Wunen and Wunen 2. J. Cell Biol. 171, 675–683.
Lippincott-Schwartz, J., Altan-Bonnet, N., and Patterson, G. H. (2003) Photobleaching and photoactivation: following protein dynamics in living cells. Nat. Cell Biol. Suppl, S7–S14.
Klonis, N., Rug, M., Harper, I., Wickham, M., Cowman, A., and Tilley L. (2002) Fluorescence photobleaching analysis for the study of cellular dynamics. Eur. Biophys. J. 31, 36–51.
Patterson, G. H. and Lippincott-Schwartz, J. (2002) A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297, 1873–1877.
Lippincott-Schwartz, J. and Patterson, G. H. (2003) Development and use of fluorescent protein markers in living cells. Science 300, 87–91.
Patterson, G. H. and Lippincott-Schwartz, J. (2004) Selective photolabeling of proteins using photoactivatable GFP. Methods 32, 445–450.
Post, J. N., Lidke, K. A., Rieger, B., and Arndt-Jovin, D. J. (2005) One-and two-photon photoactivation of a paGFP-fusion protein in live Drosophila embryos. FEBS Lett. 579, 325–330.
Gurskaya, N. G., Verkhusha, V. V., Shcheglov, A. S., et al. (2006) Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nat. Biotechnol. 24, 461–465.
Mizuno, H. Mal, T. K., Tong, K. I., et al. (2003) Photo-induced peptide cleavage in the green-to-red conversion of a fluorescent protein. Mol. Cell 12, 1051–1058.
Vincent, J. P. and O’Farrell, P. H. (1992) The state of engrailed expression is not clonally transmitted during early Drosophila development. Cell 68, 923–931.
Campos-Ortega, J. A. and Hartenstein, V. (1985) The embryonic development of Drosophila melanogaster. Springer-Verlag, Berlin, Heidelberg, New York, Tokyo.
Royou, A., Field, C., Sisson, J. C., Sullivan, W., and Karess, R. (2003) Reassessing the role and dynamics of nonmuscle myosin II during furrow formation in early Drosophila embryos. Mol. Biol. Cell 15, 838–850.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Cavey, M., Lecuit, T. (2008). Imaging Cellular and Molecular Dynamics in Live Embryos Using Fluorescent Proteins. In: Dahmann, C. (eds) Drosophila. Methods in Molecular Biology, vol 420. Humana Press. https://doi.org/10.1007/978-1-59745-583-1_13
Download citation
DOI: https://doi.org/10.1007/978-1-59745-583-1_13
Publisher Name: Humana Press
Print ISBN: 978-1-58829-817-1
Online ISBN: 978-1-59745-583-1
eBook Packages: Springer Protocols