Huebinger Jan, Masip Martin E, Christmann Jens, Wehner Frank, Bastiaens Philippe I H
Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
Faculty of Chemistry and Chemical Biology, TU Dortmund University, Dortmund, Germany.
Bio Protoc. 2017 Apr 20;7(8). doi: 10.21769/BioProtoc.2236.
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 , 2016).
通过单分子定位显微镜（SMLM）或荧光寿命成像显微镜（FLIM）进行的荧光活细胞成像原则上允许对单个活细胞中的分子模式进行时空观察。然而，细胞内分子的动态变化阻碍了对它们的精确观察。我们在此展示了一种详细的方案，用于在显微镜上对活细胞进行连续的可逆低温捕获循环，从而能够精确确定单个活细胞内分子模式的演变。通过SMLM和FLIM观察配体诱导的受体酪氨酸激酶聚集及其活性模式，证明了这种方法的有效性（马西普，2016年）。
