title: Two-photon excitation and photoconversion of EosFP in dual-color 4Pi confocal microscopy.
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: Institute of Biophysics, University of Ulm, 89069 Ulm, Germany.
authors: Sergey
authors: S
authors: Ivanchenko
authors: Sylvia
authors: S
authors: Glaschick
authors: Carlheinz
authors: C
authors: Röcker
authors: Franz
authors: F
authors: Oswald
authors: Jörg
authors: J
authors: Wiedenmann
authors: G Ulrich
authors: GU
authors: Nienhaus
mesh_terms: D008027:Light; D008164:Luminescent Proteins / Q000737:chemistry* / Q000528:radiation effects / Q000648:ultrastructure*; D018613:Microscopy, Confocal / Q000379:methods*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D010777:Photochemistry / Q000379:methods*; D011487:Protein Conformation
chemical_list: D008164:Luminescent Proteins