10.1021/acsnano.5b07595.s005 Ignacio Munguira Ignacio Munguira Ignacio Casuso Ignacio Casuso Hirohide Takahashi Hirohide Takahashi Felix Rico Felix Rico Atsushi Miyagi Atsushi Miyagi Mohamed Chami Mohamed Chami Simon Scheuring Simon Scheuring Glasslike Membrane Protein Diffusion in a Crowded Membrane American Chemical Society 2016 Glasslike Membrane Protein Diffusion document coexistence novel analysis methodology membrane diffusion plasma membrane observation capabilities Crowded MembraneMany functions protein lysenin force microscopy immobile molecules diffusion regimes time scales fluid bilayer glass phase fluorescence microscopy 2016-02-09 00:00:00 Dataset https://acs.figshare.com/articles/dataset/Glasslike_Membrane_Protein_Diffusion_in_a_Crowded_Membrane/2087782 Many functions of the plasma membrane depend critically on its structure and dynamics. Observation of anomalous diffusion <i>in vivo</i> and <i>in vitro</i> using fluorescence microscopy and single particle tracking has advanced our concept of the membrane from a homogeneous fluid bilayer with freely diffusing proteins to a highly organized crowded and clustered mosaic of lipids and proteins. Unfortunately, anomalous diffusion could not be related to local molecular details given the lack of direct and unlabeled molecular observation capabilities. Here, we use high-speed atomic force microscopy and a novel analysis methodology to analyze the pore forming protein lysenin in a highly crowded environment and document coexistence of several diffusion regimes within one membrane. We show the formation of local glassy phases, where proteins are trapped in neighbor-formed cages for time scales up to 10 s, which had not been previously experimentally reported for biological membranes. Furthermore, around solid-like patches and immobile molecules a slower glass phase is detected leading to protein trapping and creating a perimeter of decreased membrane diffusion.