Fluorescence nanoscopy by polarization modulation and polarization angle narrowing.
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.
1] Department of Biophysical Chemistry, Institute for Physical and Theoretical Chemistry, University of Braunschweig, Braunschweig, Germany. [2].
Nour
N
Hafi
1] Biomolecular Spectroscopy and Single-Molecule Detection Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany. [2].
Matthias
M
Grunwald
1] Department of Biophysical Chemistry, Institute for Physical and Theoretical Chemistry, University of Braunschweig, Braunschweig, Germany. [2].
Laura S
LS
van den Heuvel
1] Felix Bernstein Institute for Mathematical Statistics in Bioscience, Göttingen, Germany. [2] Institute for Mathematical Stochastics, University of Göttingen, Göttingen, Germany. [3] Statistical Inverse Problems in Biophysics Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany. [4].
Timo
T
Aspelmeier
Biomolecular Spectroscopy and Single-Molecule Detection Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Jian-Hua
JH
Chen
Division of Cellular Neurobiology, Zoological Institute, University of Braunschweig, Braunschweig, Germany.
Marta
M
Zagrebelsky
Institute of Organic and Biomolecular Chemistry, University of Göttingen, Göttingen, Germany.
Ole M
OM
Schütte
Institute of Organic and Biomolecular Chemistry, University of Göttingen, Göttingen, Germany.
Claudia
C
Steinem
Division of Cellular Neurobiology, Zoological Institute, University of Braunschweig, Braunschweig, Germany.
Martin
M
Korte
1] Felix Bernstein Institute for Mathematical Statistics in Bioscience, Göttingen, Germany. [2] Institute for Mathematical Stochastics, University of Göttingen, Göttingen, Germany. [3] Statistical Inverse Problems in Biophysics Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Axel
A
Munk
1] Department of Biophysical Chemistry, Institute for Physical and Theoretical Chemistry, University of Braunschweig, Braunschweig, Germany. [2] Biomolecular Spectroscopy and Single-Molecule Detection Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Peter J
PJ
Walla
D000465:Algorithms; D000818:Animals; D002478:Cells, Cultured; D003198:Computer Simulation; D004847:Epithelial Cells / Q000378:metabolism; D004867:Equipment Design; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D008870:Microtubules / Q000648:ultrastructure; D008962:Models, Theoretical; D054118:Nanospheres / Q000737:chemistry; D036103:Nanotechnology / Q000379:methods*; D016011:Normal Distribution; D017785:Photons; D048328:Potoroidae; D012984:Software
D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins