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Proceedings of the National Academy of Sciences of the United States of America v.115 no.6, 2018년, pp.1204 - 1209   SCI SCIE
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Aberration-corrected cryoimmersion light microscopy

Faoro, Raffaele (Biological Micro- and Nanotechnology, Max Planck Institute for Biophysical Chemistry, Gottingen 37073, Germany ) ; Bassu, Margherita (Biological Micro- and Nanotechnology, Max Planck Institute for Biophysical Chemistry, Gottingen 37073, Germany ) ; Mejia, Yara X. (Biological Micro- and Nanotechnology, Max Planck Institute for Biophysical Chemistry, Gottingen 37073, Germany ) ; Stephan, Till (Structure and Dynamics of Mitochondria, Max Planck Institute for Biophysical Chemistry, Gottingen 37073, Germany ) ; Dudani, Nikunj (Biological Micro- and Nanotechnology, Max Planck Institute for Biophysical Chemistry, Gottingen 37073, Germany ) ; Boeker, Christian (Carl Zeiss Microscopy GmbH, Gottingen 37081, Germany ) ; Jakobs, Stefan (Structure and Dynamics of Mitochondria, Max Planck Institute for Biophysical Chemistry, Gottingen 37073, Germany ) ; Burg, Thomas P. (Biological Micro- and Nanotechnology, Max Planck Institute for Biophysical Chemistry, Gottingen 37073, Germany; ) ;
  • 초록  

    Significance Cryofluorescence imaging is of great interest in biological microscopy because vitrified samples (i.e., frozen without ice crystallization) are free from fixation artifacts, are highly photostable, and allow direct correlation with electron cryomicroscopy. Here, we show a concept for conducting cryo-light microscopy in immersion that provides a twofold to fivefold increase in image brightness and higher resolution than are attainable with air objectives. Spherical aberration at an imaging temperature below the glass transition of water (−135 °C) is corrected by our approach. Cryogenic fluorescent light microscopy of flash-frozen cells stands out by artifact-free fixation and very little photobleaching of the fluorophores used. To attain the highest level of resolution, aberration-free immersion objectives with accurately matched immersion media are required, but both do not exist for imaging below the glass-transition temperature of water. Here, we resolve this challenge by combining a cryoimmersion medium, HFE-7200, which matches the refractive index of room-temperature water, with a technological concept in which the body of the objective and the front lens are not in thermal equilibrium. We implemented this concept by replacing the metallic front-lens mount of a standard bioimaging water immersion objective with an insulating ceramic mount heated around its perimeter. In this way, the objective metal housing can be maintained at room temperature, while creating a thermally shielded cold microenvironment around the sample and front lens. To demonstrate the range of potential applications, we show that our method can provide superior contrast in Escherichia coli and yeast cells expressing fluorescent proteins and resolve submicrometer structures in multicolor immunolabeled human bone osteosarcoma epithelial (U2OS) cells at [Formula]C.


  • 주제어

    cryo-light microscopy .   high-NA immersion objective .   fluorescence imaging .   cryofixation .   cryofluorescence microscopy.  

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