Abstract
We provide a detailed protocol for a three-dimensional long-term live imaging of cellular spheroids with light sheet-based fluorescence microscopy. The protocol allows the recording of all phases of spheroid formation in three dimensions, including cell proliferation, aggregation, and compaction. We employ the human hepatic cell line HepaRG transfected with the fusion protein H2B-GFP, i.e., a fluorescing histone. The protocol allows monitoring the effect of drugs or toxicants.
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References
Baker M (2010) Cellular imaging: taking a long, hard look. Nature 466:1137–1140
Homem CCF, Reichardt I, Berger C et al (2013) Long-term live cell imaging and automated 4D analysis of drosophila neuroblast lineages. PloS One 8(11):e79588. doi:10.1371/journal.pone.0079588
Carlton PM, Boulanger J, Kervrann C et al (2010) Fast live simultaneous multiwavelength four-dimensional optical microscopy. Proc Natl Acad Sci U S A 107:16016–16022
Huisken J, Stainier DY (2009) Selective plane illumination microscopy techniques in developmental biology. Development 136:1963–1975
Huisken J, Swoger J, Del Bene F et al (2004) Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305:1007–1009
Keller PJ, Schmidt AD, Wittbrodt J et al (2008) Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy. Science 322:1065–1069
Keller PJ, Stelzer EH (2008) Quantitative in vivo imaging of entire embryos with digital scanned laser light sheet fluorescence microscopy. Curr Opin Neurobiol 18:624–632
Maizel A, von Wangenheim D, Federici F et al (2011) High-resolution live imaging of plant growth in near physiological bright conditions using light sheet fluorescence microscopy. Plant J 68:377–385
Keller PJ, Schmidt AD, Santella A et al (2010) Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy. Nat Methods 7:637–642
Pampaloni F, Kroschewski R, Berge U et al (2014) Tissue-Culture Light Sheet Fluorescence Microscopy (TC-LSFM) allows long-term imaging of three-dimensional cell cultures under controlled conditions. Integrative Biology. doi:10.1039/C4IB00121D
Harma V, Virtanen J, Makela R et al (2010) A comprehensive panel of three-dimensional models for studies of prostate cancer growth, invasion and drug responses. PLoS One 5:e10431
Friedrich J, Seidel C, Ebner R et al (2009) Spheroid-based drug screen: considerations and practical approach. Nat Protoc 4:309–324
Matsuda M, Shiratori S (2011) Correlation of antithrombogenicity and heat treatment for layer-by-layer self-assembled polyelectrolyte films. Langmuir 27:4271–4277
Takano S, Tian W, Matsuda M et al (2011) Detection of IDH1 mutation in human gliomas: comparison of immunohistochemistry and sequencing. Brain Tumor Pathol 28:115–123
Lee MY, Kumar RA, Sukumaran SM et al (2008) Three-dimensional cellular microarray for high-throughput toxicology assays. Proc Natl Acad Sci U S A 105:59–63
Goto A, Hoshino M, Matsuda M et al (2011) Phosphorylation of STEF/Tiam2 by protein kinase A is critical for Rac1 activation and neurite outgrowth in dibutyryl cAMP-treated PC12D cells. Mol Biol Cell 22:1780–1790
Kelm JM, Timmins NE, Brown CJ et al (2003) Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnol Bioeng 83:173–180
Lin RZ, Chou LF, Chien CC et al (2006) Dynamic analysis of hepatoma spheroid formation: roles of E-cadherin and beta1-integrin. Cell Tissue Res 324:411–422
Verveer PJ, Swoger J, Pampaloni F et al (2007) High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy. Nat Methods 4:311–313
Lorenzo C, Frongia C, Jorand R et al (2011) Live cell division dynamics monitoring in 3D large spheroid tumor models using light sheet microscopy. Cell Div 6:22
Guillouzo A, Corlu A, Aninat C et al (2007) The human hepatoma HepaRG cells: a highly differentiated model for studies of liver metabolism and toxicity of xenobiotics. Chem Biol Interact 168:66–73
Greger K, Swoger J, Stelzer EH (2007) Basic building units and properties of a fluorescence single plane illumination microscope. Rev Sci Instrum 78:023705
Huisken J, Stainier DY (2007) Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM). Opt Lett 32:2608–2610
Zeng J, Du J, Lin J et al (2009) High-efficiency transient transduction of human embryonic stem cell-derived neurons with baculoviral vectors. Mol Ther 17:1585–1593
Acknowledgments
The authors thank the Deutsche Forschungsgemeinschaft (DFG) and the German Federal Ministry of Education and Research (BMBF) for financial support (project ProMEBS). We thank Berit Langer for her outstanding support.
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Pampaloni, F., Richa, R., Ansari, N., Stelzer, E.H.K. (2015). Live Spheroid Formation Recorded with Light Sheet-Based Fluorescence Microscopy. In: Verveer, P. (eds) Advanced Fluorescence Microscopy. Methods in Molecular Biology, vol 1251. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2080-8_3
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DOI: https://doi.org/10.1007/978-1-4939-2080-8_3
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2079-2
Online ISBN: 978-1-4939-2080-8
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