Time‐Lapse Imaging of Embryonic Neural Stem Cell Division in Drosophila by Two‐Photon Microscopy

Elena Rebollo1, Cayetano Gonzalez2

1 Cell Division Group, IRB‐Barcelona, Barcelona, Spain, 2 Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1H.2
DOI:  10.1002/9780470151808.sc01h02s13
Online Posting Date:  June, 2010
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This unit describes a protocol for live imaging of Drosophila embryonic neural stem cells using two‐photon microscopy. Compared to traditional one‐photon confocal imaging, this technique renders higher‐resolution optical sections from deeper within the embryo. It is ideally suited to following embryonic neuroblasts located underneath the neuroepithelial cell layer for several rounds of cell division. Curr. Protoc. Stem Cell Biol. 13:1H.2.1‐1H.2.9. © 2010 by John Wiley & Sons, Inc.

Keywords: two‐photon microscopy; live imaging; Drosophila embryo; neuroblast

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Table of Contents

  • Introduction
  • Basic Protocol 1: Time‐Lapse Imaging by Two‐Photon Microscopy
  • Support Protocol 1: Embryo Preparation
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Time‐Lapse Imaging by Two‐Photon Microscopy

  • Dechorionated embryos, mounted and ready for microscopy (see protocol 2)
  • Leica TCS‐SP5 multiphoton LSM system (or equivalent), including:
    • General components:
    • Inverted microscope (DMI 6000 CS)
    • Laser point scanner system
    • 63×/1.4 objective (oil immersion, HCX PL APO)
    • Multiphoton components:
    • Ti:sapphire laser Mai Tai (Spectra Physics) with wavelength range of 710 to 990 nm, pulse frequency of 80 MHz, giving maximum pulses of 1.5 W and 1 psec duration, and power control by EOM (electro‐optical modulator)
    • External detector (NDD, non‐descanned detection): PMT 9624 (high sensitive meshless type, selected for Leica)
    • Basic barrier filter SP720 (to stop infrared radiation and allow detection of whole GFP and YFP emission spectra)
    • Leica LAS AF acquisition software (implemented for control of infrared laser)
  • Software for visualizing and processing images (Imaris, Bitplane) and for assembling videos (Image J, http://rsb.info.nih.gov/ij/, or Adobe After Effects)

Support Protocol 1: Embryo Preparation

  • Male and female Drosophila, 1 to 2 weeks old
  • Apple plates: 3% (w/v) agar plates containing 4% (v/v) apple juice, freshly prepared and kept at 4°C
  • Freshly made yeast paste
  • Halocarbon oil (Sigma; halocarbon oil 700, cat. no. H8898 or Voltalef oil 10S)
  • Plastic fly cages pierced with small holes to allow breathing
  • Stereomicroscope
  • Fine artist's brush
  • Double‐sided cellophane tape (Scotch tape)
  • Blunt forceps, e.g., standard No. 5 dissection forceps smoothed for this purpose
  • 35‐mm culture dish: Petri dish with a coverslip bottom (e.g., Fluoro Dish sterile culture dishes, World Precision Instruments, cat. no. FD#35)
  • Teflon membranes (YSI Incorporated; cat. no. 5793)
  • Vacuum grease (Dow Corning; cat. no. 976V)
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Literature Cited

Literature Cited
   Ashburner, M. 1989. Drosophila: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Campos‐Ortega, J.A. and Hartenstein, V. 1997. The Embryonic Development of Drosophila melanogaster. Springer, Berlin.
   Cavey, M. and Lecuit, T. 2008. Imaging cellular and molecular dynamics in live embryos using fluorescent proteins. Methods Mol. Biol. 420:219‐238.
   Gonzalez, C. 2007. Spindle orientation, asymmetric division and tumour suppression in Drosophila stem cells. Nat. Rev. Genet. 8:462‐472.
   Gonzalez, C. and Glover, D.M. 2003. Techniques for studying mitosis in Drosophila. In The Cell Cycle: A Practical Approach (P. Fantes and R. Brooks, eds.), pp 143‐175. IRL Press, Oxford.
   Kaltschmidt, J.A., Davidson, C.M., Brown, N.H., and Brand, A.H. 2000. Rotation and asymmetry of the mitotic spindle direct asymmetric cell division in the developing central nervous system. Nat. Cell Biol. 2:7‐12.
   Kiehart, D.P., Crawford, J.M., and Montague, R.A. 2007. Collection, dechorionation, and preparation of Drosophila embryos for quantitative microinjection. Cold Spring Harb. Protoc. doi:10.1101/pdb.prot4717.
   Knoblich, J.A. 2008. Mechanisms of asymmetric stem cell division. Cell 132:583‐597.
   McMahon, A., Supatto, W., Fraser, S.E., and Stathopoulos, A. 2008. Dynamic analysis of Drosophila gastrulation provides insights into collective cell migration. Science 322:1546‐1550.
   Rebollo, E., Sampaio, P., Januschke, J., Llamazares, S., Varmark, H., and Gonzalez, C. 2007. Functionally unequal centrosomes drive spindle orientation in asymmetrically dividing Drosophila neural stem cells. Dev. Cell 12:467‐474.
   Rebollo, E., Roldán, M., and Gonzalez, C. 2009. Spindle alignment is achieved without rotation after the first cell cycle in Drosophila embryonic neuroblasts. Development 36:3393‐3397.
   Rothwell, W.F. and Sullivan, W. 2007. Drosophila embryo dechorionation. Cold Spring Harb. Protoc. doi:10.1101/pdb.prot4826.
   Rusan, N.M. and Peifer, M. 2007. A role for a novel centrosome cycle in asymmetric cell division. J. Cell Biol. 177:13‐20.
   Supatto, W., Debarre, D., Moulia, B., Brouzes, E., Martin, J.L., Farge, E., and Beaurepaire, E. 2005. In vivo modulation of morphogenetic movements in Drosophila embryos with femtosecond laser pulses. Proc. Natl. Acad. Sci. U.S.A. 102:1047‐1052.
   Wieschaus, E. and Nusslein‐Volhard, C. 1988. Looking at embryos. In Drosophila: A Practical Approach. (D.B. Rogers, ed.), pp. 199‐227. Oxford University Press, Oxford.
   Williams, D.W. and Truman, J.W. 2005. Cellular mechanisms of dendrite pruning in Drosophila: Insights from in vivo time‐lapse of remodeling dendritic arborizing sensory neurons. Development 132:3631‐3642.
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