title: Reversible cryo-arrest for imaging molecules in living cells at high spatial resolution.
abstract: 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.
authors.affiliation: Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
authors.forename: Martin E
authors.initials: ME
authors.lastname: Masip
authors.affiliation: Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Huebinger
authors.affiliation: Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.|Faculty of Chemistry and Chemical Biology, TU Dortmund, Dortmund, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Christmann
authors.affiliation: Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
authors.forename: Ola
authors.initials: O
authors.lastname: Sabet
authors.affiliation: Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Wehner
authors.affiliation: Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
authors.forename: Antonios
authors.initials: A
authors.lastname: Konitsiotis
authors.affiliation: Fraunhofer Institute for Biomedical Engineering, St. Ingbert, Germany.
authors.forename: Günther R
authors.initials: GR
authors.lastname: Fuhr
authors.affiliation: Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.|Faculty of Chemistry and Chemical Biology, TU Dortmund, Dortmund, Germany.
authors.forename: Philippe I H
authors.initials: PIH
authors.lastname: Bastiaens
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D003080:Cold Temperature; D003451:Cryoprotective Agents / Q000737:chemistry*; D011992:Endosomes / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D010766:Phosphorylation; D036104:Receptor, EphA2 / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D003451:Cryoprotective Agents; C512478:EGFR protein, human; D066246:ErbB Receptors; D036104:Receptor, EphA2