Reversible Cryo-arrests of Living Cells to Pause Molecular Movements for High-resolution Imaging
Jan Huebinger,
Martin Masip,
Jens Christmann,
Frank Wehner,
Philippe Bastiaens
Affiliations
Jan Huebinger
Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany
Martin Masip
Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany
Jens Christmann
Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, GermanyFaculty of Chemistry and Chemical Biology, TU Dortmund University, Dortmund, Germany
Frank Wehner
Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany
Philippe Bastiaens
Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, GermanyFaculty of Chemistry and Chemical Biology, TU Dortmund University, Dortmund, Germany
Fluorescence live-cell imaging by single molecule localization microscopy (SMLM) or fluorescence lifetime imaging microscopy (FLIM) in principle allows for the spatio-temporal observation of molecular patterns in individual, living cells. However, the dynamics of molecules within cells hamper their precise observation. We present here a detailed protocol for consecutive cycles of reversible cryo-arrest of living cells on a microscope that allows for a precise determination of the evolution of molecular patterns within individual living cells. The usefulness of this approach has been demonstrated by observing ligand-induced clustering of receptor tyrosine kinases as well as their activity patterns by SMLM and FLIM (Masip et al., 2016).
