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Fluorescence Localization After Photobleaching (FLAP)

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

¹The Randall Division, King's College London, London, United Kingdom
²Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, London, United Kingdom

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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Abstract

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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Unit Introduction
Basic Protocol: 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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Materials

Basic Protocol: FLAP of Actin in Living Cells

Materials

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--actin and eYFP--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 Support Protocol 1 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. 21.2.1)
Software:
- Zeiss LSM 510 operating software for image acquisition
- Zeiss LSM Reader for image review (free download; see Internet Resources)

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 Support Protocol 1)

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% CO₂ incubator unless otherwise specified. Some media (e.g., DMEM) require altered levels of CO₂ to maintain pH 7.4.

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

Materials

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

Materials

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

Looking for materials? Suppliers Guide

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Figures

Figure 21.2.1

Zeiss upright LSM 510 microscope contained within a 37°C environmental control incubator. In the authors' laboratory, the enviromental control chamber is a specially built Plexiglass box with access hatches (Cancer Research UK workshop). The essential components for the heater and control system are a centrifugal fan (part no. 40BTFL from Air Flow Developments), and a temperature controller (208-2739), thermocouple type T (219-4680), box (584-615), mini-3-pole plug (449-269), mini-3-pole socket (449-275), fuse holder (418-603), optical relay (394-535), type T panel socket (219-4860), type T line plug (219-4876), enclosed heater (224-565), and thermocouple connector (219-4876), all from RS Components. A similar commercially available system (Microscope Temperature Control System) may be obtained from Life Imaging Services (see Internet Resources).

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Figure 21.2.2

Window from Zeiss LSM software showing four channels and fifth combined channel after setting gains and offsets and laser powers. Courtesy of Carl Zeiss, Germany; reprinted with permission of Zeiss UK.

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Figure 21.2.3

Window from Zeiss LSM software showing only the combined channel after bleaching during data recording. Courtesy of Carl Zeiss, Germany; reprinted with permission of Zeiss U K.

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Figure 21.2.4

Window from Zeiss LSM software showing custom Hall pseudocolor palette. Note that the lowest intensity level is coded as black and the highest as white. Courtesy of Carl Zeiss, Germany; reprinted with permission of Zeiss UK.

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Figure 21.2.5

Window from Zeiss LSM software showing both tracks on the configuration panel. Courtesy of Carl Zeiss, Germany; reprinted with permission of Zeiss UK.

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Figure 21.2.6

(A) The summed intensity values for a whole cell during a 10-min time series after step in Support Protocol 2. These values have been normalized so that the total intensity of the first CFP image is 1. (B) The same intensity values after fade compensation as in step of Support Protocol 2. This consists of dividing each CFP image by a factor so that the total intensity is 1 and then dividing the corresponding YFP image by the same factor. Note that fluctuations due to cyclical focus drift have also been compensated.

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Figure 21.2.7

Absolute FLAP (A, C) and relative FLAP (B, D) images of the cell featured in Figures 21.2.2 and 21.2.3 recorded immediately after bleaching (A, B) and 3.9 sec later (C, D). Bleach box is shown as white rectangle. Hall palette.

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Literature Cited

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
	http://www.lis.ch
Web site of Life Imaging Services, which supplies microscope temperature control systems.
	http://www.zeiss.com/us/micro/home.nsf/Contents-FrameDHTML/286BA4D22B14D...
Zeiss Web site from which LSM Reader can be downloaded.
	http://www.sciencemag.org/cgi/content/full/300/5616/142/DC1
Supplementary online material for Zicha et al. (2003). Describes diffusion modeling.