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Two-photon excitation and photoconversion of EosFP in dual-color 4Pi confocal microscopy.
Biophys J. 2007 Jun 15; 92(12):4451-7.BJ

Abstract

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.

Authors+Show Affiliations

Institute of Biophysics, University of Ulm, 89069 Ulm, Germany.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

17384061

Citation

Ivanchenko, Sergey, et al. "Two-photon Excitation and Photoconversion of EosFP in Dual-color 4Pi Confocal Microscopy." Biophysical Journal, vol. 92, no. 12, 2007, pp. 4451-7.
Ivanchenko S, Glaschick S, Röcker C, et al. Two-photon excitation and photoconversion of EosFP in dual-color 4Pi confocal microscopy. Biophys J. 2007;92(12):4451-7.
Ivanchenko, S., Glaschick, S., Röcker, C., Oswald, F., Wiedenmann, J., & Nienhaus, G. U. (2007). Two-photon excitation and photoconversion of EosFP in dual-color 4Pi confocal microscopy. Biophysical Journal, 92(12), 4451-7.
Ivanchenko S, et al. Two-photon Excitation and Photoconversion of EosFP in Dual-color 4Pi Confocal Microscopy. Biophys J. 2007 Jun 15;92(12):4451-7. PubMed PMID: 17384061.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Two-photon excitation and photoconversion of EosFP in dual-color 4Pi confocal microscopy. AU - Ivanchenko,Sergey, AU - Glaschick,Sylvia, AU - Röcker,Carlheinz, AU - Oswald,Franz, AU - Wiedenmann,Jörg, AU - Nienhaus,G Ulrich, Y1 - 2007/03/23/ PY - 2007/3/27/pubmed PY - 2007/7/27/medline PY - 2007/3/27/entrez SP - 4451 EP - 7 JF - Biophysical journal JO - Biophys. J. VL - 92 IS - 12 N2 - 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. SN - 0006-3495 UR - https://www.unboundmedicine.com/medline/citation/17384061/Two_photon_excitation_and_photoconversion_of_EosFP_in_dual_color_4Pi_confocal_microscopy_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0006-3495(07)71245-6 DB - PRIME DP - Unbound Medicine ER -