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The positive switching fluorescent protein Padron2 enables live-cell reversible saturable optical linear fluorescence transitions (RESOLFT) nanoscopy without sequential illumination steps

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Konen,  T.
Department of NanoBiophotonics, MPI for Biophysical Chemistry, Max Planck Society;

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Stumpf,  D.
Department of NanoBiophotonics, MPI for Biophysical Chemistry, Max Planck Society;

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Grotjohann,  T.
Department of NanoBiophotonics, MPI for biophysical chemistry, Max Planck Society;

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Jansen,  I.
Department of NanoBiophotonics, MPI for Biophysical Chemistry, Max Planck Society;

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Weber,  M.
Department of NanoBiophotonics, MPI for Biophysical Chemistry, Max Planck Society;

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Jensen,  N. A.
Department of NanoBiophotonics, MPI for Biophysical Chemistry, Max Planck Society;

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Hell,  S. W.
Department of NanoBiophotonics, MPI for Biophysical Chemistry, Max Planck Society;

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Jakobs,  S.
Research Group of Mitochondrial Structure and Dynamics, MPI for biophysical chemistry, Max Planck Society;

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Citation

Konen, T., Stumpf, D., Grotjohann, T., Jansen, I., Bossi, M., Weber, M., et al. (2021). The positive switching fluorescent protein Padron2 enables live-cell reversible saturable optical linear fluorescence transitions (RESOLFT) nanoscopy without sequential illumination steps. ACS Nano, 15(6), 9509-9521. doi:10.1021/acsnano.0c08207.


Cite as: http://hdl.handle.net/21.11116/0000-0009-6569-0
Abstract
Reversibly switchable fluorescent proteins (RSFPs) can be repeatedly transferred between a fluorescent on- and a nonfluorescent off-state by illumination with light of different wavelengths. Negative switching RSFPs are switched from the on- to the off-state with the same wavelength that also excites fluorescence. Positive switching RSFPs have a reversed light response, where the fluorescence excitation wavelength induces the transition from the off- to the on-state. Reversible saturable optical linear (fluorescence) transitions (RESOLFT) nanoscopy utilizes these switching states to achieve diffraction-unlimited resolution but so far has primarily relied on negative switching RSFPs by using time sequential switching schemes. On the basis of the green fluorescent RSFP Padron, we engineered the positive switching RSFP Padron2. Compared to its predecessor, it can undergo 50-fold more switching cycles while displaying a contrast ratio between the on- and the off-states of more than 100:1. Because of its robust switching behavior, Padron2 supports a RESOLFT imaging scheme that entirely refrains from sequential switching as it only requires beam scanning of two spatially overlaid light distributions. Using Padron2, we demonstrate live-cell RESOLFT nanoscopy without sequential illumination steps.