488 nm laser

Confocal microscopy images were taken on a FV-1000/IX81 confocal laser scanning microscope equipped with GaAsP detectors, an UPlanSApo ×60/1.20 objective and a 488 nm laser (Olympus, Tokyo, Japan). Exposure time was set to 2 µs/pixel, the laser was set to 500 hv with a gain of 1 and 3 % offset.

Localization and tracking of single molecules were done as previously described^{16 (link)}. Briefly, infected HeLa LAMP1-GFP or RAW264.7 cells were labeled with the HaloTag ligand coupled to tetramethylrhodamine (HTL-TMR, Promega G8251) in a concentration of 20 nM for 15 min at 37 °C. After 10 washing steps, the cells were imaged with an imaging medium consisting of Minimal Essential Medium (MEM) with Earle’s salts, without NaHCO₃, without L-Glutamine, without phenol red (Merck D1145) and supplemented with 30 mM HEPES, pH 7.4. TIRF microscopy was performed using an inverted microscope Olympus IX-81, equipped with an incubation chamber maintaining 37 °C and humidity, a motorized 4-line TIRF condenser, a ×150 objective (UAPON 150x TIRF, NA 1.45), a TIRF quadband polychroic mirror (zt405/488/561/640rpc), a 488 nm laser (150 mW, Olympus), and a 561 nm laser (150 mW, Olympus). Localization, as well as tracking of single molecules, were carried out with the help of a self-written user interface in MatLab 2013a^{21 ,46 (link)–50 (link)}.