Reversible cryo-arrest for imaging molecules in living cells at high spatial resolution
| Autor: | Ola Sabet, Jan Huebinger, Antonios D. Konitsiotis, Philippe I. H. Bastiaens, Günther R Fuhr, Jens Christmann, Frank Wehner, Martin E. Masip |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Fluorescence-lifetime imaging microscopy Cell signaling Endosomes Biochemistry Article Receptor tyrosine kinase 03 medical and health sciences Cryoprotective Agents Microscopy Humans Epidermal growth factor receptor Phosphorylation Molecular Biology biology Chemistry Super-resolution microscopy Receptor EphA2 Cell Membrane Cell Biology Molecular Imaging Cell biology Cold Temperature ErbB Receptors 030104 developmental biology Microscopy Fluorescence biology.protein Biophysics Signal transduction Molecular imaging HeLa Cells Signal Transduction Biotechnology |
| Zdroj: | Nature Methods. 13:665-672 |
| ISSN: | 1548-7105 1548-7091 |
| DOI: | 10.1038/nmeth.3921 |
| Popis: | The dynamics of molecules in living cells hampers precise imaging of molecular patterns by functional and super-resolution microscopy. We developed a method that circumvents lethal chemical fixation and allows on-stage cryo-arrest for consecutive imaging of molecular patterns within the same living, but arrested, cells. The reversibility of consecutive cryo-arrests was demonstrated by the high survival rate of different cell lines and by intact growth factor signaling that was not perturbed by stress response. Reversible cryo-arrest was applied to study the evolution of ligand-induced receptor tyrosine kinase activation at different scales. The nanoscale clustering of epidermal growth factor receptor (EGFR) in the plasma membrane was assessed by single-molecule localization microscopy, and endosomal microscale activity patterns of ephrin receptor A2 (EphA2) were assessed by fluorescence lifetime imaging microscopy. Reversible cryo-arrest allows the precise determination of molecular patterns while conserving the dynamic capabilities of living cells. |
| Databáze: | OpenAIRE |
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