title: Three-dimensional locations of gold-labeled proteins in a whole mount eukaryotic cell obtained with 3nm precision using aberration-corrected scanning transmission electron microscopy.
abstract: Three-dimensional (3D) maps of proteins within the context of whole cells are important for investigating cellular function. However, 3D reconstructions of whole cells are challenging to obtain using conventional transmission electron microscopy (TEM). We describe a methodology to determine the 3D locations of proteins labeled with gold nanoparticles on whole eukaryotic cells. The epidermal growth factor receptors on COS7 cells were labeled with gold nanoparticles, and critical-point dried whole-mount cell samples were prepared. 3D focal series were obtained with aberration-corrected scanning transmission electron microscopy (STEM), without tilting the specimen. The axial resolution was improved with deconvolution. The vertical locations of the nanoparticles in a whole-mount cell were determined with a precision of 3nm. From the analysis of the variation of the axial positions of the labels we concluded that the cellular surface was ruffled. To achieve sufficient stability of the sample under electron beam irradiation during the recording of the focal series, the sample was carbon coated. A quantitative method was developed to analyze the stability of the ultrastructure after electron beam irradiation using TEM. The results of this study demonstrate the feasibility of using aberration-corrected STEM to study the 3D nanoparticle distribution in whole cells.
authors: Department of Chemistry, Vanderbilt University, Nashville, TN 37235, USA.
authors: Madeline J
authors: MJ
authors: Dukes
authors: Ranjan
authors: R
authors: Ramachandra
authors: Jean-Pierre
authors: JP
authors: Baudoin
authors: W
authors: W
authors: Gray Jerome
authors: Niels
authors: N
authors: de Jonge
mesh_terms: D000818:Animals; D019556:COS Cells / Q000648:ultrastructure; D002522:Chlorocebus aethiops; D005057:Eukaryotic Cells; D006046:Gold / Q000737:chemistry*; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D017348:Microscopy, Electron, Scanning Transmission / Q000379:methods*; D053758:Nanoparticles / Q000737:chemistry / Q000648:ultrastructure; D011506:Proteins / Q000737:chemistry*
chemical_list: D011506:Proteins; D006046:Gold