Fluorescence Imaging Techniques for Studying Drosophila Embryo Development

Manos Mavrakis1, Richa Rikhy2, Mary Lilly2, Jennifer Lippincott‐Schwartz2

1 Institute of Developmental Biology of Marseille‐Luminy, UMR6216 CNRS‐Université de la Méditerranée, Marseille, France, 2 Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.18
DOI:  10.1002/0471143030.cb0418s39
Online Posting Date:  June, 2008
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Abstract

This unit describes fluorescence‐based techniques for noninvasive imaging of development in living Drosophila embryos, discussing considerations for fluorescent imaging within living embryos and providing protocols for generation of flies expressing fluorescently tagged proteins and for preparation of embryos for fluorescent imaging. The unit details time‐lapse confocal imaging of live embryos and discusses optimizing image acquisition and performing three‐dimensional imaging. Finally, the unit provides a variety of specific methods for optical highlighting of specific subsets of fluorescently tagged proteins and organelles in the embryo, including fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), and photoactivation techniques, permitting analysis of specific movements of fluorescently tagged proteins within cells. These protocols, together with the relative ease of generating transgenic animals and the ability to express tagged proteins in specific tissues or at specific developmental times, provide powerful means for examining in vivo behavior of any tagged protein in embryos in myriad mutant backgrounds. Curr. Protoc. Cell Biol. 39:4.18.1‐4.18.43. © 2008 by John Wiley & Sons, Inc.

Keywords: Drosophila; embryo; fluorescence; imaging; photobleaching; photoactivation

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Generation of Transgenic Drosophila for Live Fluorescence Microscopy Using the Gal4/Uas System
  • Basic Protocol 2: Preparation of Drosophila Embryos for Fluorescence Microscopy
  • Basic Protocol 3: Time‐Lapse Confocal Imaging of Living Drosophila Embryos
  • Basic Protocol 4: Time‐Lapse Imaging of Living Drosophila Embryos with Two‐Photon Laser Scanning microscopy
  • Basic Protocol 5: Fluorescence Recovery after Photobleaching in Living Drosophila Embryos Using a Laser Scanning Confocal Microscope Capable of Selective Photobleaching
  • Basic Protocol 6: Fluorescence Loss in Photobleaching in Living Drosophila Embryos Using a Laser Scanning Confocal Microscope Capable of Selective Photobleaching
  • Basic Protocol 7: Photoactivation in Living Drosophila Embryos Using a Laser Scanning Confocal Microscope Capable of Selective Photobleaching
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Generation of Transgenic Drosophila for Live Fluorescence Microscopy Using the Gal4/Uas System

  Materials
  • Gene of interest cDNA: e.g., from Drosophila Genomics Research Center (DGRC), http://dgrc.cgb.indiana.edu
  • Vector carrying genetically encoded fluorescent protein (FP) variant (e.g., mCherry, Clontech; EGFP and Cerulean, Addgene)
  • pUASP or pUAST vector for subcloning FP‐tagged gene of interest and for transforming Drosophila (DGRC)
  • Drosophila Gal4 line for driving expression in tissue of interest (see FlyBase, http://flystocks.bio.indiana.edu/Browse/misc‐browse/gal4.htm)
  • Drosophila balancer stock for generating flies for dual fluorescent imaging: Sp/SM6a; MKRS, Sb/TM6, Tb (see FlyBase, http://flystocks.bio.indiana.edu/Browse/balancers/bala ncers.htm)
  • Stereomicroscope with epifluorescence illumination (e.g., Zeiss Stemi SV11 or Leica MZ FLIII, with 1.0 or 1.6 objectives and 10 eyepiece)
  • Additional reagents and equipment for cloning genes (see appendix 3A)

Basic Protocol 2: Preparation of Drosophila Embryos for Fluorescence Microscopy

  Materials
  • Embryo collection cages that fit 60‐mm petri dishes (Genesee Scientific)
  • Apple or grape juice agar plates (see recipe)
  • Yeast paste: prepared by mixing 1 g dry yeast with 1.8 ml distilled H 2O to make a thick paste
  • 50% bleach: bleach (4% to 6% available chlorine) diluted 1:1 with distilled H 2O just prior to use
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Dichlorodimethylsilane (Fluka)
  • Egg basket (Fig. B): prepared by removing the stem of a plastic funnel (∼60 mm top internal diameter) with a blade, slightly melting the small diameter opening, and attaching to a piece of 100‐µm stainless steel wire or 100‐ or 120‐µm nylon mesh (Genesee Scientific 57‐102 or BD Falcon 352360)
  • Squirt bottle with distilled H 2O
  • Dissecting microscope (e.g., Zeiss Stemi SV6/11 or Stemi 2000)
  • Fine paintbrush (No. 1) for transferring and handling embryos
  • Imaging chamber: No. 1.0 Borosilicate LabTek Chambered Coverglass (Nunc) or No. 1.0 Borosilicate MatTek Glass‐Bottom Dishes (MatTek)

Basic Protocol 3: Time‐Lapse Confocal Imaging of Living Drosophila Embryos

  Materials
  • Drosophila embryos expressing fluorescent proteins (FPs) of interest ( protocol 1), mounted in LabTek or MatTek imaging chamber ( protocol 2)
  • Confocal laser scanning microscope (CLSM; e.g., Zeiss LSM 510 or Olympus FluoView FV1000) equipped with:
    • Laser lines and filter sets appropriate for the FPs of interest
    • High‐numerical‐aperture (NA), water‐immersion objective (e.g., Zeiss C‐Apochromat 40×/1.20 W Corr UV‐VIS‐IR water lens or Olympus UPLAPO 60×/1.2 NA water lens)

Basic Protocol 4: Time‐Lapse Imaging of Living Drosophila Embryos with Two‐Photon Laser Scanning microscopy

  Materials
  • Drosophila embryos expressing fluorescent proteins (FPs) of interest ( protocol 1), mounted in LabTek or MatTek imaging chamber ( protocol 2)
  • Zeiss LSM 510 META equipped with:
    • Coherent Chameleon titanium/sapphire laser, tunable from 720 nm to 930 nm
    • Filter sets appropriate for the FPs of interest
    • High‐numerical‐aperture (NA) water‐immersion objective (e.g., Zeiss C‐Apochromat 40×/1.20 W Corr UV‐VIS‐IR water lens)

Basic Protocol 5: Fluorescence Recovery after Photobleaching in Living Drosophila Embryos Using a Laser Scanning Confocal Microscope Capable of Selective Photobleaching

  Materials
  • Drosophila embryos expressing fluorescent proteins (FPs) of interest ( protocol 1), mounted in LabTek or MatTek imaging chamber ( protocol 2)
  • Confocal laser scanning microscope (CLSM) capable of selective photobleaching (e.g., Zeiss LSM 510 or Olympus FluoView FV1000), equipped with laser lines and filter sets appropriate for the FPs of interest

Basic Protocol 6: Fluorescence Loss in Photobleaching in Living Drosophila Embryos Using a Laser Scanning Confocal Microscope Capable of Selective Photobleaching

  Materials
  • Drosophila embryos expressing fluorescent proteins (FPs) of interest ( protocol 1), mounted in LabTek or MatTek imaging chamber ( protocol 2)
  • Confocal laser scanning microscope capable of selective photobleaching (e.g., Zeiss LSM 510 or Olympus FluoView FV1000), equipped with laser lines and filter sets appropriate for the FPs of interest

Basic Protocol 7: Photoactivation in Living Drosophila Embryos Using a Laser Scanning Confocal Microscope Capable of Selective Photobleaching

  Materials
  • Drosophila embryos expressing PA‐GFP‐ or mEosFP‐tagged proteins of interest (see protocol 1), mounted in LabTek or MatTek imaging chamber (see protocol 2)
  • Confocal laser scanning microscope capable of selective photobleaching (e.g., Zeiss LSM 510 or Olympus FluoView FV1000), equipped with a violet light laser line (e.g., a 405‐nm diode laser or a 413‐nm krypton laser), as well as visible light lasers appropriate for imaging PA‐GFP or mEosFP
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Figures

Videos

Literature Cited

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