Proem emccd camera

DNA origami was imaged below the diffraction limit of light via DNA-PAINT^{17 (link)} using an inverted Nikon Eclipse Ti2 microscope from Nikon Instruments in total internal reflectance fluorescence (TIRF) mode. The images were acquired using: an optical feedback focal-drift correction system developed in-house or the Perfect Focus System from Nikon Instruments; an oil-immersion CFI Apochromat ×100 TIRF objective with a 1.49 numerical aperture, plus an extra ×1.5 magnification from Nikon Instruments; and a 405/488/561/647 nm Laser Quad Band Set TIRF filter cube from Chroma. A 561 nm laser source excited fluorescence from the DNA-PAINT imager strands within an evanescent field extending a few hundred nanometers above the surface of the glass coverslip. The emitted fluorescence was imaged onto the full chip with 512 × 512 pixels (1 pixel = 16 μm) using a ProEM EMCCD camera from Princeton Instruments at a 300 ms exposure time (~3 frames/s). During an experimental recording, each of the individual data strands, within a dNAM origami’s matrix, transiently and repeatedly bound an imager strand, which emits a signal, creating a series of blinks. Images with blinking events were recorded into a stack (typically 40,000 frames per recording) using Nikon NIS-Elements version 5.20.00 (Nikon Instruments) or LightField version 5 (Princeton Instruments) prior to processing and analysis.

LentiBrite^™ Paxillin (Millipore)-GFP-tagged FAK Y397E and FAK Y397F cells were plated (10,000 per well) in regular growth medium in Lab-Tek II chambered coverglass (Nunc) coated with 10 μg/ml of fibronectin (Calbiochem). The focal adhesion turnover analysis was performed on a custom-made system (Visitron Systems) based on an Nikon Eclipse Ti inverted microscope, an Nikon 60x 1.40 NA objective and a ProEM EMCCD camera (Princeton Instruments) in an incubation chamber (Oxolab) to control temperature, CO2 (5% premixed) and humidity. Images were acquired every three minutes for four hours using VisiView software (Visitron Systems) and analyzed by ImageJ.