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  Achromatic light patterning and improved image reconstruction for parallelized RESOLFT nanoscopy.

Chmyrov, A., Leutenegger, M., Grotjohann, T., Schönle, A., Keller-Findeisen, J., Kastrup, L., et al. (2017). Achromatic light patterning and improved image reconstruction for parallelized RESOLFT nanoscopy. Scientific Reports, 7: 44619. doi:10.1038/srep44619.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-002C-DECA-8 Version Permalink: http://hdl.handle.net/21.11116/0000-0002-A70C-5
Genre: Journal Article

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 Creators:
Chmyrov, A., Author
Leutenegger, M.1, Author              
Grotjohann, T.1, Author              
Schönle, A.1, Author              
Keller-Findeisen, J.1, Author              
Kastrup, L., Author
Jakobs, S.2, Author              
Donnert, G., Author
Sahl, S. J.1, Author              
Hell, S. W.1, Author              
Affiliations:
1Department of NanoBiophotonics, MPI for biophysical chemistry, Max Planck Society, ou_578627              
2Research Group of Mitochondrial Structure and Dynamics, MPI for biophysical chemistry, Max Planck Society, ou_578566              

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 Abstract: Fluorescence microscopy is rapidly turning into nanoscopy. Among the various nanoscopy methods, the STED/RESOLFT super-resolution family has recently been expanded to image even large fields of view within a few seconds. This advance relies on using light patterns featuring substantial arrays of intensity minima for discerning features by switching their fluorophores between ‘on’ and ‘off’ states of fluorescence. Here we show that splitting the light with a grating and recombining it in the focal plane of the objective lens renders arrays of minima with wavelength-independent periodicity. This colour-independent creation of periodic patterns facilitates coaligned on- and off-switching and readout with combinations chosen from a range of wavelengths. Applying up to three such periodic patterns on the switchable fluorescent proteins Dreiklang and rsCherryRev1.4, we demonstrate highly parallelized, multicolour RESOLFT nanoscopy in living cells for ~100 × 100 μm2 fields of view. Individual keratin filaments were rendered at a FWHM of ~60–80 nm, with effective resolution for the filaments of ~80–100 nm. We discuss the impact of novel image reconstruction algorithms featuring background elimination by spatial bandpass filtering, as well as strategies that incorporate complete image formation models.

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Language(s): eng - English
 Dates: 2017-03-20
 Publication Status: Published online
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 Rev. Method: Internal
 Identifiers: DOI: 10.1038/srep44619
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Title: Scientific Reports
Source Genre: Journal
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Pages: 9 Volume / Issue: 7 Sequence Number: 44619 Start / End Page: - Identifier: -