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Localized multiphoton photoactivation of paGFP in Drosophila wing imaginal discs

Pantazis, Periklis and González-Gaitán, Marcos (2007) Localized multiphoton photoactivation of paGFP in Drosophila wing imaginal discs. Journal of Biomedical Optics, 12 (4). Art. No. 044004. ISSN 1083-3668. doi:10.1117/1.2770478.

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In biological imaging of fluorescent molecules, multiphoton laser scanning microscopy (MPLSM) has become the favorite method of fluorescence microscopy in tissue explants and living animals. The great power of MPLSM with pulsed lasers in the infrared wavelength lies in its relatively deep optical penetration and reduced ability to cause potential nonspecific phototoxicity. These properties are of crucial importance for long time-lapse imaging. Since the excited area is intrinsically confined to the high-intensity focal volume of the illuminating beam, MPLSM can also be applied as a tool for selectively manipulating fluorophores in a known, three-dimensionally defined volume within the tissue. Here we introduce localized multiphoton photoactivation (MP-PA) as a technique suitable for analyzing the dynamics of photoactivated molecules with three-dimensional spatial resolution of a few micrometers. Short, intense laser light pulses uncage photoactivatable molecules via multiphoton excitation in a defined volume. MP-PA is demonstrated on photoactivatable paGFP in Drosophila wing imaginal discs. This technique is especially useful for extracting quantitative information about the properties of photoactivatable fusion proteins in different cellular locations in living tissue as well as to label single or small patches of cells in tissue to track their subsequent lineage.

Item Type:Article
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Pantazis, Periklis0000-0002-8367-9332
Additional Information:© 2007 Society of Photo-Optical Instrumentation Engineers. Paper 06280RR received Oct. 6, 2006; revised manuscript received Mar. 15, 2007; accepted for publication Mar. 16, 2007; published online Aug. 23, 2007. We thank G. Patterson and J. Lippincott-Schwartz who kindly provided the paGFP-plasmid. We thank S.E. Fraser, A. Oates, V. Dudu, and N. Pantazis for critical reading and comments on the manuscript. We are indebted to K. Anderson and J. Peychl for assistance at the Biorad Radiance 2100 MP setup. We thank D. Backasch and A. Schwabedissen for technical assistance. This work was supported by the Max Planck Society, DFG, VW, and HFSP.
Funding AgencyGrant Number
Max Planck SocietyUNSPECIFIED
Deutsche Forschungsgemeinschaft (DFG)UNSPECIFIED
Volkswagen StiftungUNSPECIFIED
Human Frontier Science ProgramUNSPECIFIED
Subject Keywords:green fluorescent protein; photoactivation; two-photon microscopy; Drosophila wing imaginal disc.
Issue or Number:4
Record Number:CaltechAUTHORS:20161025-132609922
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Official Citation:Periklis Pantazis and Marcos González-Gaitán "Localized multiphoton photoactivation of paGFP in Drosophila wing imaginal discs", J. Biomed. Opt. 12(4), 044004 (August 23, 2007). ;
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:71464
Deposited By: George Porter
Deposited On:25 Oct 2016 21:19
Last Modified:11 Nov 2021 04:45

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