Na oil uplsapo objective

The Olympus Fluoview™ FV1000 confocal microscope was equipped with a temperature controlled CO₂ incubation chamber at 37°C (EMBL, Heidelberg, Germany) and a 60x/1.35 NA Oil UPLSApo objective (Olympus, Hamburg, Germany). Fluorescent fusion proteins with BFP, mCitrine and mCherry or Alexa647-, Alexa555-, Alexa488-labeled secondary antibodies were excited using the 405 nm Diode-UV laser (FV5-LD05, Hatagaya), 488 nm Argon-laser (GLG 3135, Showa Optronics, Tokyo, Japan), 561 nm DPSS laser (85-YCA-020-230, Melles Griot, Bensheim, Germany) and 633 nm HeNe laser (05LHP-991, Melles Griot, Bensheim, Germany), respectively. Detection of fluorescence emission was restricted with an Acousto-Optical Beam Splitter (AOBS) as follows: BFP (425-–478 nm), mCitrine/Alexa488 (498-–551 nm), mCherry/Alexa555 (575-–675 nm), Alexa647 (655-–755 nm). In all cases, scanning was performed in frame-by-frame sequential mode with 2x frame averaging. The pinhole was set between 1.5 and 2.5 airy units.