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Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy


Fluorescent proteins that can be reversibly photoswitched between a fluorescent and a nonfluorescent state are important for innovative microscopy schemes, such as protein tracking1, fluorescence resonance energy transfer imaging2, sub-diffraction resolution microscopy3,4,5,6,7,8,9 and others. However, all available monomeric reversibly switchable fluorescent proteins (RSFPs) have similar properties and switching characteristics10,11,12, thereby limiting their use. Here, we introduce two bright green fluorescent RSFPs, bsDronpa and Padron, generated by extensive mutagenesis of the RSFP Dronpa10, with unique absorption and switching characteristics. Whereas bsDronpa features a broad absorption spectrum extending into the UV, Padron displays a switching behavior that is reversed to that of all green fluorescent RSFPs known to date. These two RSFPs enable live-cell fluorescence microscopy with multiple labels using a single detection color, because they can be distinguished by photoswitching. Furthermore, we demonstrate dual-color fluorescence microscopy with sub-diffraction resolution using bsDronpa and Dronpa whose emission maxima are separated by <20 nm.

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Figure 1: Properties of rsFastLime, Padron and bsDronpa.
Figure 2: Monochromatic triple-label imaging in living E. coli cells.
Figure 3: 3D confocal time-lapse imaging of living budding yeast cells expressing rsFastLime and Padron using reversible switching to discriminate between the fluorescence signals from the two proteins.
Figure 4: Dual-color, single-molecule-switching-based nanoscopy image of cryosections of E. coli cells expressing membrane-anchored bsDronpa or Dronpa.


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We thank S. Löbermann and J. Schilde for excellent technical assistance and J. Jethwa for carefully reading the manuscript. We acknowledge J.-W.L. de Gier, Stockholm University, Sweden, for providing us with the expression vector harboring the M13-GFP fusion.

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Authors and Affiliations



M.A. performed experiments, analyzed data and wrote the manuscript. A.C.S., J.F. and D.W. performed experiments. A.S. and A.E. provided algorithms and software for sub-diffraction resolution microscopy. C.E. constructed the fluorescence microscope utilized for screening. S.W.H. designed research and wrote the manuscript. S.J. analyzed data, designed research and wrote the manuscript; all authors contributed to editing the manuscript.

Corresponding author

Correspondence to Stefan Jakobs.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11, Tables 1–3, Methods (PDF 1936 kb)

Supplementary Movie 1

3D confocal time lapse imaging of live cells expressing rsFastLime and Padron (MOV 209 kb)

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Andresen, M., Stiel, A., Fölling, J. et al. Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy. Nat Biotechnol 26, 1035–1040 (2008).

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