Eclipse t1 microscope

For kinetic measurement of secretory trafficking in living cells, HeLa cells were grown in Lap-Tek chambers and transfected with Str-Ii_VSVGwt-SBP-EGFP (Boncompain et al., 2012 (link)). These were mounted in a chamber with 5% CO₂ at 37°C on an Visitron spinning disk Nikon Eclipse T1 microscope with a 60X, 1.4 NA, CFI, Plan Apo λ Oil objective. Positions for imaging were marked and a pre-release image was acquired. Release of proteins for targeting to the Golgi complex was induced by addition of 250 μM biotin. Time-lapse imaging was performed for 15 min, acquiring images over z-stacks every 1 min. Integrated pixel intensities at the Golgi and in the whole cell were calculated using ImageJ. Fluorescence intensities were normalized relative to the fraction at the Golgi complex before biotin addition.

Confocal fluorescence images were acquired with LSM 880, LSM 780 microscope (ZEISS) or a Leica SP2 AOBS microscope. For all microscopes, 63 × 1.4 NA, oil plan-apochromat objectives were used. For time-lapse microscopy, cells were seeded into μ-slide 4-well ibidi chambers and incubated with 0.1 μM SiR-Hoechst 1 hr prior to the experiment. Images were acquired at a Visitron spinning disk Nikon Eclipse T1 microscope, using a 60 × 1.4 oil lens, in a 5% CO₂, 37°C chamber. 15 z-stacks were acquired every 5 min for 8 to 12 hr.