Movie 8

Continuous imaging permits simultaneous acquisition of actin cytoskeleton and adhesion dynamics. CHO-K1 cells stably expressing paxillin-EGFP were transfected with LifeAct-mRuby and seeded onto fibronectin-coated dishes. Cells were simultaneously illuminated for 20 mins with a 491 nm laser set to 0.016 mW to image EGFP and a 561 nm laser set to 0.031 mW to image mRuby. Images were captured with an HCX PL APO 63x/1.40NA oil objective and camera exposure time of 5 s. 2x2 pixel binning was used to enhance S/N. EGFP and mRuby emission signals were sent to two separate sCMOS cameras using a dichroic mirror, then colour coded and digitally combined. Focus drift affected brightness and contrast in the images. Time is in min:s. Scale bar is 5 μm.

Optimizing live-cell fluorescence imaging conditions to minimize phototoxicity

Alex Kiepas, Elena Voorand, Firas Mubaid, Peter M. Siegel, and Claire M. Brown

J Cell Sci 2020. 133:None-None; doi: 10.1242/jcs.242834