Fluorescence Localization After Photobleaching (FLAP)

Graham A. Dunn1, Mark R. Holt1, Daniel Y. H. Soong1, Colin Gray2, Daniel Zicha2

1 The Randall Division, King's College London, London, 2 Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, London
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 21.2
DOI:  10.1002/0471143030.cb2102s24
Online Posting Date:  October, 2004
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Fluorescence localization after photobleaching is a new method for localized photolabeling and subsequent tracking of specific molecules within living cells. The molecular species to be located carries two different fluorophores that can be imaged independently but simultaneously by fluorescence microscopy. For the method to work, these two fluorophores should be accurately colocalized throughout the cell so that their images are closely matched. One of the fluorophores (the target fluorophore) is then rapidly photobleached at a chosen location. The unbleached (reference) fluorophore remains colocalized with the target fluorophore; thus, the subsequent fate of the photobleached molecules can be revealed by processing simultaneously acquired digital images of the two fluorophores. Here we demonstrate the simplicity and effectiveness of the FLAP method in revealing both fast and slow molecular dynamics in living cells using a Zeiss LSM 510 laser scanning confocal microscope.

Keywords: Fluorescence microscopy; laser scanning confocal microscopy; FLAP; GFP; photobleaching; actin; cell motility

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Table of Contents

  • Basic Protocol 1: FLAP of Actin in Living Cells
  • Support Protocol 1: Setting up the LSM 510 and its Software
  • Support Protocol 2: Image Processing and Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: FLAP of Actin in Living Cells

  • Rat fibroblast cell line K2 or T15
  • Hanks' Minimal Essential Medium (MEM; Cancer Research UK; daniel.zicha@cancer.org.uk) containing 10% bovine serum and no antibiotics
  • cDNA constructs of eCFP‐β‐actin and eYFP‐β‐actin (see recipe)
  • Experimental reagents of interest (e.g., myosin light chain kinase inhibitor, ML‐7)
  • Hot wax mixture (see recipe)
  • Non‐toxic immersion oil optimized for 37°C, refractive index 1.515 (Cargille Labs)
  • 18 × 18–mm glass coverslips
  • 35‐mm plastic petri dishes (Costar)
  • Microinjection system (also see units 4.10 & 17.1) including:
    • 5171 micromanipulator (Eppendorf)
    • 5246 transjector (Eppendorf)
  • Zeiss Axiovert 35 microscope
  • Microneedles (GC120TF‐10, Harvard Apparatus)
  • P97 Flaming/Brown micropipette puller (Sutter)
  • Optical chambers (see recipe)
  • Zeiss upright LSM 510 microscope (see protocol 2 for full configuration) contained within a 37°C environmental control incubator (e.g., Microscope Temperature Control System, Life Imaging Services) or a similar apparatus assembled in house (Fig. )
  • Software:
    • Zeiss LSM 510 operating software for image acquisition
    • Zeiss LSM Reader for image review (free download; see )
  • Additional reagents and equipment for cell culture (unit 1.1), microinjection (see units 4.10 & 17.1), and use of LSM 510 operating software (see 21.2)
NOTE: All solutions and equipment coming into contact with living cells must be sterile and aseptic technique should be used accordingly.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) require altered levels of CO 2 to maintain pH 7.4.

Support Protocol 1: Setting up the LSM 510 and its Software

  • Zeiss upright LSM 510 microscope and software (see Basic Protocol 1)
  • Small beads (e.g., TetraSpeck microspheres, 0.2 µm; Molecular Probes)

Support Protocol 2: Image Processing and Analysis

  • Mathematica v 4.2 or 5 (Wolfram Research) or a dedicated image‐processing package capable of processing 12‐bit TIFF images
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Literature Cited

   Choidas, A., Jungbluth, A., Sechi, A., Murphy, J., Ullrich, A., and Marriott, G. 1998. The suitability and application of a GFP‐actin fusion protein for long‐term imaging of the organization and dynamics of the cytoskeleton in mammalian cells. Eur. J. Cell Biol. 77:81‐90.
   Chudakov, D.M., Belousov, V.V., Zaraisky, A.G., Novoselov, V.V., Staroverov, D.B., Zorov, D.B., Lukyanov, S., and Lukyanov, K.A. 2003. Kindling fluorescent proteins for precise in vivo photolabeling. Nat. Biotechnol. 2:191‐194.
   Dunn, G.A., Dobbie, I.M., Monypenny J, Holt, MR., and Zicha, D. 2002. Fluorescence Localization After Photobleaching (FLAP): A new method for studying protein dynamics in living cells. J. Micros. 205:109‐112.
   Holt, M.R., Soong, D.Y.H., Monypenny J., Dobbie, I.M., Zicha, D., and Dunn, G.A. 2004. Using bioprobes to follow protein dynamics in living cells. In Cell Motility: From Molecules to Organisms, Chapter 7 (A. Ridley, P. Clark, and M. Peckham, eds.) John Wiley and Sons, Hoboken, N.J..
   Lippincott‐Schwartz, J., Snapp, E., and Kenworthy, A. 2001. Studying protein dynamics in living cells. Nat. Rev. Mol. Cell Biol. 2:444‐456.
   McGrath, J.L., Tardy, Y., Dewey, C.F., Jr, Meister, J.J., and Hartwig, J.H. 1998. Simultaneous measurements of actin filament turnover, filament fraction, and monomer diffusion in endothelial cells. Biophys. J. 75:2070‐2078.
   Mitchison, T.J., Sawin, K.E., Theriot, J.A., Gee, K., and Mallavarapu, A. 1998. Caged fluorescent probes. Methods Enzymol. 291:63‐78.
   Patterson G.H. and Lippincott‐Schwartz, J. 2002. A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297:1873‐1877.
   Watanabe, N. and Mitchison, T.J. 2002. Single‐molecule speckle analysis of actin filament turnover in lamellipodia. Science 295:1083‐1086.
   Waterman‐Storer, C.M. and Salmon, E.D. 1997. Actomyosin‐based retrograde flow of microtubules in the lamella of migrating epithelial cells influences microtubule dynamic instability and turnover and is associated with microtubule breakage and treadmilling. J. Cell Biol. 139:417‐434.
   Zicha, D., Dobbie, I.M., Holt, MR., Monypenny J., Soong, D.Y.H., Gray, C., and Dunn, G.A. 2003. Rapid actin transport during cell protrusion. Science 300:142‐145.
Key References
   Dunn et al., 2002. See above.
  First description of the technique.
   Zicha et al., 2003. See above.
  Describes an application of the technique.
Internet Resources
  Web site of Life Imaging Services, which supplies microscope temperature control systems.
  Zeiss Web site from which LSM Reader can be downloaded.
  Supplementary online material for Zicha et al. (). Describes diffusion modeling.
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