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Reversible cryo-arrest for imaging molecules in living cells at high spatial resolution

: Masip, M.E.; Huebinger, J.; Christmann, J.; Sabet, O.; Wehner, F.; Konitsiotis, A.; Fuhr, G.R.; Bastiaens, P.I.H.


Nature methods 13 (2016), Nr.8, S.665-672
ISSN: 1548-7091
ISSN: 1548-7105
Fraunhofer IBMT ()

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.