Abstract
Light sheet fluorescent microscopy (LSFM), and in particular its most widespread flavor Selective Plane Illumination Microscopy (SPIM), promises to provide unprecedented insights into developmental dynamics of entire living systems. By combining minimal photo-damage with high imaging speed and sample mounting tailored toward the needs of the specimen, it enables in toto imaging of embryogenesis with high spatial and temporal resolution. Drosophila embryos are particularly well suited for SPIM imaging because the volume of the embryo does not change from the single cell embryo to the hatching larva. SPIM microscopes can therefore image Drosophila embryos embedded in rigid media, such as agarose, from multiple angles every few minutes from the blastoderm stage until hatching. Here, we describe sample mounting strategies to achieve such a recording. We also provide detailed protocols to realize multiview, long-term, time-lapse recording of Drosophila embryos expressing fluorescent markers on the commercially available Zeiss Lightsheet Z.1 microscope and the OpenSPIM.
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References
Huisken J, Swoger J, Del Bene F, Wittbrodt J, Stelzer EH (2004) Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science, 305(5686):1007–1009.
Keller PJ (2013) Imaging morphogenesis: technological advances and biological insights. Science, 340(6137):1234168. doi:10.1126/science.1234168
Krzic U, Gunther S, Saunders TE, Streichan SJ, Hufnagel L (2012) Multiview light-sheet microscope for rapid in toto imaging. Nat Methods, 9(7):730–3. doi:10.1038/nmeth.2064
Tomer R, Khairy K, Amat F, Keller PJ (2012) Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy. Nat Methods, 9(7):755–63. doi:10.1038/nmeth.2062
Ejsmont RK, Sarov M, Winkler S, Lipinski KA, Tomancak P (2009) A toolkit for high-throughput, cross-species gene engineering in Drosophila. Nat Methods, 6(6):435–7. doi:10.1038/nmeth.1334
Nagarkar-Jaiswal S, DeLuca SZ, Lee P-T, Lin W-W, Pan H, Zuo Z, Lv J, Spradling AC, Bellen HJ (2015) A genetic toolkit for tagging intronic MiMIC containing genes. eLife, 4. doi:10.7554/eLife.08469
Reynaud EG, Krzic U, Greger K, Stelzer EHK (2008) Light sheet‐based fluorescence microscopy: more dimensions, more photons, and less photodamage. HFSP J, 2(5):266–75. doi:10.2976/1.2974980.
Schmied C, Stamataki E, Tomancak P (2014) Open-source solutions for SPIMage processing. Methods Cell Biol, 123:505–29. doi:10.1016/B978-0-12-420138-5.00027-6
Preibisch S, Saalfeld S, Schindelin J, Tomancak P (2010) Software for bead-based registration of selective plane illumination microscopy data. Nat Methods, 7(6):418-9. doi:10.1038/nmeth0610-418.
Preibisch S, Amat F, Stamataki E, Sarov M, Singer RH, Myers E, Tomancak P (2014) Efficient Bayesian-based multiview deconvolution. Nat Methods, 11(6):645–8. doi:10.1038/nmeth.2929
Pitrone PG, Schindelin J, Stuyvenberg L, Preibisch S, Weber M, Eliceiri KW, Huisken J, Tomancak P (2013) OpenSPIM: an open-access light-sheet microscopy platform. Nat Methods , 10(7):598–9. doi:10.1038/nmeth.2507
Huisken J, Stainier DYR (2007) Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM). Opt Lett, 1;32(17):2608–10.
Preibisch S, Saalfeld S (2009) Bead-based mosaicing of single plane illumination microscopy images using geometric local descriptor matching. SPIE Medical Imaging, 7259, 72592S–725102S. doi:10.1117/12.812612
Weber M, Mickoleit M, Huisken J (2014) Light sheet microscopy. Methods Cell Biol, 123:193–215. doi:10.1016/B978-0-12-420138-5.00011-2
Gao L, Shao L, Chen B-C, Betzig E (2014) 3D live fluorescence imaging of cellular dynamics using Bessel beam plane illumination microscopy. Nat Protoc, 9(5):1083–101. doi:10.1038/nprot.2014.087
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Schmied, C., Tomancak, P. (2016). Sample Preparation and Mounting of Drosophila Embryos for Multiview Light Sheet Microscopy. In: Dahmann, C. (eds) Drosophila. Methods in Molecular Biology, vol 1478. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6371-3_10
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DOI: https://doi.org/10.1007/978-1-4939-6371-3_10
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