Live-cell multiplane three-dimensional super-resolution optical fluctuation imaging

Stefan Geissbuehler (), Azat Sharipov, Aurélien Godinat, Noelia L. Bocchio, Patrick A. Sandoz, Anja Huss, Nickels A. Jensen, Stefan Jakobs, Jörg Enderlein, F. Gisou van der Goot, Elena A. Dubikovskaya, Theo Lasser and Marcel Leutenegger
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Stefan Geissbuehler: École Polytechnique Fédérale de Lausanne, Laboratoire d’Optique Biomédicale
Azat Sharipov: École Polytechnique Fédérale de Lausanne, Laboratoire d’Optique Biomédicale
Aurélien Godinat: École Polytechnique Fédérale de Lausanne, Laboratory of Bioorganic Chemistry and Molecular Imaging, Institute of Chemical Sciences and Engineering (ISIC)
Noelia L. Bocchio: École Polytechnique Fédérale de Lausanne, Laboratoire d’Optique Biomédicale
Patrick A. Sandoz: École Polytechnique Fédérale de Lausanne, Global Health Institute
Anja Huss: Georg August University, III. Institute of Physics
Nickels A. Jensen: Max Planck Institute for Biophysical Chemistry
Stefan Jakobs: Max Planck Institute for Biophysical Chemistry
Jörg Enderlein: Georg August University, III. Institute of Physics
F. Gisou van der Goot: École Polytechnique Fédérale de Lausanne, Global Health Institute
Elena A. Dubikovskaya: École Polytechnique Fédérale de Lausanne, Laboratory of Bioorganic Chemistry and Molecular Imaging, Institute of Chemical Sciences and Engineering (ISIC)
Theo Lasser: École Polytechnique Fédérale de Lausanne, Laboratoire d’Optique Biomédicale
Marcel Leutenegger: École Polytechnique Fédérale de Lausanne, Laboratoire d’Optique Biomédicale

Nature Communications, 2014, vol. 5, issue 1, 1-7

Abstract: Abstract Super-resolution optical fluctuation imaging (SOFI) provides an elegant way of overcoming the diffraction limit in all three spatial dimensions by computing higher-order cumulants of image sequences of blinking fluorophores acquired with a classical widefield microscope. Previously, three-dimensional (3D) SOFI has been demonstrated by sequential imaging of multiple depth positions. Here we introduce a multiplexed imaging scheme for the simultaneous acquisition of multiple focal planes. Using 3D cross-cumulants, we show that the depth sampling can be increased. The simultaneous acquisition of multiple focal planes significantly reduces the acquisition time and thus the photobleaching. We demonstrate multiplane 3D SOFI by imaging fluorescently labelled cells over an imaged volume of up to 65 × 65 × 3.5 μm3 without depth scanning. In particular, we image the 3D network of mitochondria in fixed C2C12 cells immunostained with Alexa 647 fluorophores and the 3D vimentin structure in living Hela cells expressing the fluorescent protein Dreiklang.

Date: 2014
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DOI: 10.1038/ncomms6830

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