Photoconvertible Behavior of LSSmOrange Applicable for Single Emission Band Optical Highlighting.
Photoswitchable fluorescent proteins are capable of changing their spectral properties upon light irradiation, thus allowing one to follow a chosen subpopulation of molecules in a biological system. Recently, we revealed a photoinduced absorption band shift of LSSmOrange, which was originally engineered to have a large energy gap between excitation and emission bands. Here, we evaluated the performance of LSSmOrange as a fluorescent tracer in living cells. The absorption maximum of LSSmOrange in HeLa cells shifted from 437 nm to 553 nm upon illumination with a 405-, 445-, 458-, or 488-nm laser on a laser-scanning microscope, whereas the emission band remained same (∼570 nm). LSSmOrange behaves as a freely diffusing protein in living cells, enabling the use of the protein as a fluorescence tag for studies of protein dynamics. By targeting LSSmOrange in mitochondria, we observed an exchange of soluble molecules between the matrices upon mitochondrial fusion. Since converted and unconverted LSSmOrange proteins have similar emission spectra, this tracer offers unique possibilities for multicolor imaging. The fluorescence emission from LSSmOrange was spectrally distinguishable from that of eYFP and mRFP, and could be separated completely by applying linear unmixing. Furthermore, by using a femtosecond laser at 850 nm, we showed that a two-photon process could evoke a light-induced red shift of the absorption band of LSSmOrange, providing a strict confinement of the conversion volume in a three-dimensional space.
Laboratory of Biomolecular Network Dynamics, Biochemistry, Molecular and Structural Biology Section, KU Leuven, Heverlee, Belgium.
Herlinde
H
De Keersmaecker
Laboratory of Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Heverlee, Belgium.
Eduard
E
Fron
Laboratory of Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Heverlee, Belgium.
Susana
S
Rocha
Cell Function Dynamics, Brain Science Institute, RIKEN, Saitama, Japan.
Takako
T
Kogure
Cell Function Dynamics, Brain Science Institute, RIKEN, Saitama, Japan.
Atsushi
A
Miyawaki
Laboratory of Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Heverlee, Belgium.
Johan
J
Hofkens
Laboratory of Biomolecular Network Dynamics, Biochemistry, Molecular and Structural Biology Section, KU Leuven, Heverlee, Belgium. Electronic address: hideaki.mizuno@kuleuven.be.
Hideaki
H
Mizuno
D000180:Acrylic Resins; D004058:Diffusion; D004926:Escherichia coli; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism; D063154:Mitochondrial Dynamics / Q000502:physiology; D061848:Optical Imaging; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D000180:Acrylic Resins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C016680:polyacrylamide gels