Live-cell multiplane three-dimensional super-resolution optical fluctuation imaging
| Autor: | F. Gisou van der Goot, Marcel Leutenegger, Theo Lasser, Azat Sharipov, Elena A. Dubikovskaya, Stefan Geissbuehler, Stefan Jakobs, Anja Huss, Aurélien Godinat, Patrick A. Sandoz, Noelia L. Bocchio, Nickels A. Jensen, Joerg Enderlein |
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| Rok vydání: | 2014 |
| Předmět: |
Diffraction
Microscope Green Fluorescent Proteins General Physics and Astronomy 02 engineering and technology cumulant imaging Article General Biochemistry Genetics and Molecular Biology Cell Line law.invention Myoblasts Mice 03 medical and health sciences Imaging Three-Dimensional Optics Sampling (signal processing) law Microscopy Animals Humans Vimentin Fluorescent protein fluorescence nanoscopy Fluorescent Dyes 030304 developmental biology Physics 0303 health sciences Multidisciplinary business.industry General Chemistry Carbocyanines 021001 nanoscience & nanotechnology Photobleaching Superresolution Mitochondria Molecular Imaging Microscopy Fluorescence Acquisition time 0210 nano-technology business HeLa Cells |
| Zdroj: | Nature Communications |
| ISSN: | 2041-1723 |
| DOI: | 10.1038/ncomms6830 |
| Popis: | 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. Super-resolution optical fluctuation imaging provides 3D images of biological specimens via blinking fluorophores. Geissbuehler et al. present a multiplexed version of this method that captures images at multiple focal planes simultaneously, reducing the acquisition time compared with standard approaches. |
| Databáze: | OpenAIRE |
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