Ixon back illuminated emccd camera

For single-molecule tracking and IF imaging for ITGB1 and paxillin, the TIRFM was performed using a Nikon Eclipse TE2000 inverted microscope with a × 100/1.49NA Plan Apo objective (Nikon). Samples were illuminated by 430, 488, and 561 nm lines of solid-state lasers (Andor Technology) and images were acquired by the iXon back-illuminated EMCCD camera (Andor Technology). The OptoSplit II Image Splitter (Cairn research) was used to simultaneously record images with two different spectral windows. For confocal imaging for 3D HER2 location patterns, 3D spheroid assays and IF imaging for E-cadherin and ZO-1, we used a Yokogawa CSU-X1 Spinning Disk Unit (Andor Technology) with an iXon DU-897-BV monochrome CCD (Andor Technology). The TIRFM and the spinning disk confocal units were installed at the ports on the opposite sides of the inverted microscope to allow for switching between two different image modes during visualization of the same cells. The imaging environment was maintained at 5% CO₂, 37 °C. Tracking of individual molecules and cells and image rendering were performed using Imaris (Bitplane) or Image-J (NIH). The cluster analysis was created in Matlab (Mathworks).