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Live Imaging of Cellular Internalization of Single Colloidal Particle by Combined Label-Free and Fluorescence Total Internal Reflection Microscopy
Version 2 2015-12-24, 18:36
Version 1 2015-11-09, 15:51
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posted on 2015-12-24, 18:36 authored by Gerard
D. Byrne, Driton Vllasaliu, Franco H. Falcone, Michael G. Somekh, Snjezana StolnikIn
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.
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Single Colloidal Particlediameter model polystyrene colloid associatescolloid internalizationTIRF arm imagestransfected 3 T 3 fibroblasts cells1 μ mparticle size limitLive ImagingInternal Reflection MicroscopyInCellular Internalizationparticle endocytosisTIRM armmembrane eventsclathrin endocytosis pathway500 nmcell structurecell membrane featuresreflectance fluorescencereflection microscopyparticle interactiondiameter colloids
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