title: Live Imaging of Cellular Internalization of Single Colloidal Particle by Combined Label-Free and Fluorescence Total Internal Reflection Microscopy.
abstract: In this work we utilize the combination of label-free total internal reflection microscopy and total internal reflectance fluorescence (TIRM/TIRF) microscopy to achieve a simultaneous, live imaging of single, label-free colloidal particle endocytosis by individual cells. The TIRM arm of the microscope enables label free imaging of the colloid and cell membrane features, while the TIRF arm images the dynamics of fluorescent-labeled clathrin (protein involved in endocytosis via clathrin pathway), expressed in transfected 3T3 fibroblasts cells. Using a model polymeric colloid and cells with a fluorescently tagged clathrin endocytosis pathway, we demonstrate that wide field TIRM/TIRF coimaging enables live visualization of the process of colloidal particle interaction with the labeled cell structure, which is valuable for discerning the membrane events and route of colloid internalization by the cell. We further show that 500 nm in diameter model polystyrene colloid associates with clathrin, prior to and during its cellular internalization. This association is not apparent with larger, 1 μm in diameter colloids, indicating an upper particle size limit for clathrin-mediated endocytosis.
authors.affiliation: Drug Delivery and Tissue Engineering Division, School of Pharmacy, University of Nottingham , Nottingham, NG7 2RD, U.K.|Institute of Biophysics, Imaging and Optical Science (IBIOS), School of Electrical and Electronic Engineering, University of Nottingham , Nottingham, NG7 2RD, U.K.
authors.forename: Gerard D
authors.initials: GD
authors.lastname: Byrne
authors.affiliation: Drug Delivery and Tissue Engineering Division, School of Pharmacy, University of Nottingham , Nottingham, NG7 2RD, U.K.
authors.forename: Driton
authors.initials: D
authors.lastname: Vllasaliu
authors.affiliation: Division of Molecular and Cellular Science, School of Pharmacy, University of Nottingham , Nottingham, NG7 2RD, U.K.
authors.forename: Franco H
authors.initials: FH
authors.lastname: Falcone
authors.affiliation: Institute of Biophysics, Imaging and Optical Science (IBIOS), School of Electrical and Electronic Engineering, University of Nottingham , Nottingham, NG7 2RD, U.K.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Somekh
authors.affiliation: Drug Delivery and Tissue Engineering Division, School of Pharmacy, University of Nottingham , Nottingham, NG7 2RD, U.K.
authors.forename: Snjezana
authors.initials: S
authors.lastname: Stolnik
mesh_terms: D016475:3T3 Cells; D000818:Animals; D003102:Colloids / Q000737:chemistry*; D005347:Fibroblasts / Q000166:cytology* / Q000648:ultrastructure*; D005453:Fluorescence; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D051379:Mice; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence / Q000379:methods*; D011108:Polymers / Q000737:chemistry*
chemical_list: D003102:Colloids; D011108:Polymers
keywords: clathrin; endocytosis; evanescent wave microscopy; imaging systems; live cell imaging; total internal reflection fluorescence