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Journal Article

Fluorescence nanoscopy by polarization modulation and polarization angle narrowing.

MPS-Authors
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Grunwald,  M.
Research Group of Biomolecular Spectroscopy and Single-Molecule Detection, MPI for biophysical chemistry, Max Planck Society;

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Aspelmeier,  T.
Research Group of Statistical Inverse-Problems in Biophysics, MPI for Biophysical Chemistry, Max Planck Society;

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Chen,  J.
Research Group of Biomolecular Spectroscopy and Single-Molecule Detection, MPI for biophysical chemistry, Max Planck Society;

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Munk,  A.
Research Group of Statistical Inverse-Problems in Biophysics, MPI for Biophysical Chemistry, Max Planck Society;

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Walla,  P. J.
Research Group of Biomolecular Spectroscopy and Single-Molecule Detection, MPI for biophysical chemistry, Max Planck Society;

External Ressource
Fulltext (public)

2032678.pdf
(Publisher version), 2MB

Supplementary Material (public)

2032678_Suppl_1.pdf
(Supplementary material), 945KB

2032678_Suppl_2.zip
(Supplementary material), 7MB

2032678_Suppl_3.pdf
(Supplementary material), 89KB

Citation

Hafi, N., Grunwald, M., van den Heuvel, L. S., Aspelmeier, T., Chen, J., Zagrebelsky, M., et al. (2014). Fluorescence nanoscopy by polarization modulation and polarization angle narrowing. Nature Methods, 11(5), 579-584. doi:10.1038/NMETH.2919.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0019-B811-0
Abstract
When excited with rotating linear polarized light, differently oriented fluorescent dyes emit periodic signals peaking at different times. We show that measurement of the average orientation of fluorescent dyes attached to rigid sample structures mapped to regularly defined (50 nm)(2) image nanoareas can provide subdiffraction resolution (super resolution by polarization demodulation, SPoD). Because the polarization angle range for effective excitation of an oriented molecule is rather broad and unspecific, we narrowed this range by simultaneous irradiation with a second, de-excitation, beam possessing a polarization perpendicular to the excitation beam (excitation polarization angle narrowing, ExPAN). This shortened the periodic emission flashes, allowing better discrimination between molecules or nanoareas. Our method requires neither the generation of nanometric interference structures nor the use of switchable or blinking fluorescent probes. We applied the method to standard wide-field microscopy with camera detection and to two-photon scanning microscopy, imaging the fine structural details of neuronal spines.