Ultra du 897u cs0 bv

Samples were mounted in a buffer consisting of 90% glycerol and 10% 10xPBS with an overall concentration of 10mM cysteamine and 50mM sodium sulfite (J.T. Baker 3922–01). Images were recorded on a modified Nikon Ti-E inverted microscope using a 60× 1.49 N.A. oil immersion objective using an EMCCD camera (Andor IXon Ultra DU-897U-CS0-#BV). The objective was mounted on a custom mount to suppress thermal and mechanical drift and focusing was provided by a piezoelectric objective positioner (Physik Instrumente P-725.4CA). For illumination a 642nm solid-state laser (Omicron Lux, 20 kW/cm2 at the sample) was used with a dichroic (Semrock, Di02-R635-25×36) and a band-pass filter (Semrock, BLP01-647R-25) for separation of excitation and emission. Field of view were selected using metal halide lamp (Prior Lumen 200) illumination before switching to laser illumination and were then imaged for approximately 20,000 frames using an exposure time of 25 ms and an EM-multiplication setting corresponding to a gain of 35.5. Events were detected and localized (Baddeley et al., 2009 (link)) and reconstructions were rendered using jittered triangulation (Baddeley et al., 2010 (link)) (see supplemental information).