Planapo λ100x 1.45 na objective lens

Live HUVEC monolayers expressing GFP-RLC and labeled with VE-Cad Dylight 550 were imaged using a Nikon A1R laser scanning confocal microscope with a PlanApo λ100X 1.45 NA objective lens (see above). At least two pre-bleach images of invasions or normal stress fibers were acquired. Photobleaching of GFP- RLC was performed for 5 iterations with 100% 488 nm laser transmission at 1.9 μs pixel dwell. We have conducted numerous prior experiments to confirm that our photobleaching condition will photobleach the targeted zone when observed with maximal pinhole size. Image acquisitions of fluorescence recovery were performed 15–20 seconds following initial FRAP for 5 min to obtain more data points within the linear range of the recovery curve to increase confidence of our k-value calculations. Imaging intervals were then increased to every 45 seconds to assure complete recovery following photobleaching. The intensity within the bleached region and a non-bleached stress fiber (as a control) were first measured using Fiji/ImageJ and then plotted with the FRAPCalc program (Dr. Rolf Sara, Åbo Akademi, Turku, Finland) to obtain the mobile fraction, t_1/2, and k values. Statistical significance between TCIA and stress fibers was determined using an unpaired t-test.

Laser scanning confocal images were captured using Nikon Elements software on a Nikon A1R confocal microscope using a PlanApo λ100X 1.45 NA objective lens (Nikon). Parameters were set to satisfy the Nyquist criterion. Spinning disk confocal microscopy was performed on an Andor XDI Revolution (Andor Technology). The cells were imaged with a Nikon Plan Apo 100×1.49NA TIRF objective every 4 minutes. Images were acquired using MetaMorph with an Andor Neo cMOS camera.