Live Cell Imaging with Widefield Microscopy

Two wide-field imaging systems were used to capture images of live cells: a Nikon Ti-E-based system and an Olympus cellSens. Details of our live imaging protocols were described in Karanasios et al., 2013 (link), Karanasios et al., 2016 (link). Briefly, cells were plated onto 60 mm dishes and transiently transfected with the relevant plasmids. After 24 hr, cells were replated onto 22-mm-diameter glass coverslips and used for imaging on the following morning. Throughout live imaging, cells were maintained at 37°C in a full enclosure incubation system. The Nikon Ti-E-based system comprised a Nikon Ti-E microscope, 100x 1.4 N.A. objective (Nikon), SpecraX LED illuminator (Lumencor), 410/504/582/669-Di01 and Di01-R442/514/561 dichroic mirrors (Semrock), Hamamatsu Flash 4.0 sCMOS camera, emission filter wheel (Sutter Instruments) and was controlled using Nikon Elements software. The Olympus cellSens system comprised of Olympus IX83 microscope, 100x 1.49 N.A. objective (Olympus), pE-4000 LED illuminator (CoolLED), Hamamatsu Flash 4.0 sCMOS camera, ZT440-445/488-491-594 dichroic mirror (Chroma), Olympus filter wheels on excitation and emission paths and was controlled using Olympus cellSens software.

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Zachari M., Gudmundsson S.R., Li Z., Manifava M., Shah R., Smith M., Stronge J., Karanasios E., Piunti C., Kishi-Itakura C., Vihinen H., Jokitalo E., Guan J.L., Buss F., Smith A.M., Walker S.A., Eskelinen E.L, & Ktistakis N.T. (2019). Selective Autophagy of Mitochondria on a Ubiquitin-Endoplasmic-Reticulum Platform. Developmental Cell, 50(5), 627-643.e5.

Publication 2019

cellSens Nikon Ti-E microscope SpecraX LED illuminator 410/504/582/669-Di01 and Di01-R442/514/561 dichroic mirrors Hamamatsu Flash 4.0 sCMOS camera Olympus IX83 microscope

Cells Dishes Microscope Plasmids

Corresponding Organization : Babraham Institute

Other organizations : University of Helsinki, University of Cambridge, University of Cincinnati Medical Center, University College London

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Variable analysis

independent variables

Transfection with relevant plasmids

dependent variables

Live cell imaging

control variables

Cells plated onto 60 mm dishes
Cells replated onto 22-mm-diameter glass coverslips
Cells maintained at 37°C in a full enclosure incubation system
Nikon Ti-E-based system: Nikon Ti-E microscope, 100x 1.4 N.A. objective, SpecraX LED illuminator, dichroic mirrors, Hamamatsu Flash 4.0 sCMOS camera, emission filter wheel, Nikon Elements software
Olympus cellSens system: Olympus IX83 microscope, 100x 1.49 N.A. objective, pE-4000 LED illuminator, Hamamatsu Flash 4.0 sCMOS camera, dichroic mirror, Olympus filter wheels, Olympus cellSens software

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