This Article Full Text Full Text (PDF) Supplement All Versions of this Article: biophysj.106.103408v1 92/12/4451    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ivanchenko, S. Articles by Nienhaus, G. U. Search for Related Content PubMed PubMed Citation Articles by Ivanchenko, S. Articles by Nienhaus, G. U.

Two-Photon Excitation and Photoconversion of EosFP in Dual-Color 4Pi Confocal Microscopy

Sergey Ivanchenko ^*, Sylvia Glaschick ^*, Carlheinz Röcker ^*, Franz Oswald , Jörg Wiedenmann  and G. Ulrich Nienhaus ^* 

^* Institute of Biophysics, Department of Internal Medicine I, and Institute of General Zoology and Endocrinology, University of Ulm, 89069 Ulm, Germany; and Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Correspondence: Address reprint requests to Gerd Ulrich Nienhaus, University of Ulm, Institute of Biophysics, 89069 Ulm, Germany. Tel.: 49-731-50-23050; Fax: 49-731-50-23059; E-mail: uli{at}uiuc.edu.

Recent years have witnessed enormous advances in fluorescence microscopy instrumentation and fluorescent marker development. 4Pi confocal microscopy with two-photon excitation features excellent optical sectioning in the axial direction, with a resolution in the 100 nm range. Here we apply this technique to cellular imaging with EosFP, a photoactivatable autofluorescent protein whose fluorescence emission wavelength can be switched from green (516 nm) to red (581 nm) by irradiation with 400-nm light. We have measured the two-photon excitation spectra and cross sections of the green and the red species as well as the spectral dependence of two-photon conversion. The data reveal that two-photon excitation and photoactivation of the green form of EosFP can be selectively performed by choosing the proper wavelengths. Optical highlighting of small subcellular compartments was shown on HeLa cells expressing EosFP fused to a mitochondrial targeting signal. After three-dimensionally confined two-photon conversion of EosFP within the mitochondrial networks of the cells, the converted regions could be resolved in a 3D reconstruction from a dual-color 4Pi image stack.

This article has been cited by other articles:

				A. Arkhipov, J. Huve, M. Kahms, R. Peters, and K. Schulten Continuous Fluorescence Microphotolysis and Correlation Spectroscopy Using 4Pi Microscopy Biophys. J., December 1, 2007; 93(11): 4006 - 4017. [Abstract] [Full Text] [PDF]