Multiplane 3D superresolution optical fluctuation imaging

نویسندگان

  • Stefan Geissbuehler
  • Azat Sharipov
  • Aurélien Godinat
  • Noelia L. Bocchio
  • Elena A. Dubikovskaya
  • Theo Lasser
  • Marcel Leutenegger
چکیده

By switching fluorophores on and off in either a deterministic [1, 2] or a stochastic [3–7] manner, superresolution microscopy has enabled the imaging of biological structures at resolutions well beyond the diffraction limit. Superresolution optical fluctuation imaging (SOFI) [7, 8] 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 conventional widefield microscope. So far, three-dimensional (3D) SOFI has only been demonstrated by sequential imaging of multiple depth positions [9, 10]. Here we introduce a versatile imaging scheme which allows for the simultaneous acquisition of multiple focal planes. Using 3D cross-cumulants, we show that the depth sampling can be increased. Consequently, the simultaneous acquisition of multiple focal planes reduces the acquisition time and hence the photo-bleaching of fluorescent markers. We demonstrate multiplane 3D SOFI by imaging the mitochondria network in fixed C2C12 cells over a total volume of 65 × 65 × 3.5 μm without depth scanning.

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تاریخ انتشار 2013