Ff01 609 54 25

Aspergillus nidulans expressing the genetically-encoded Ca²⁺ biosensor R-GECO was imaged with a motorized fluorescence stereo microscope (SMZ-25; Nikon) equipped with P2-SHR PLAN APO 1x/0.16 objective lens (Nikon) and an sCMOS camera (ORCA-Flash4.0 V2; Hamamatsu Photonics) (13 (link), 53 ). R-GECO was excited using a mercury lamp (Intensilight Hg Illuminator; Nikon), a 561/14-nm excitation filter (FF01-561/14-25, Semrock), and a 575-nm dichroic mirror (Di02-R561-25 × 36; Semrock). The red fluorescent signal passing through a 609/54-nm filter (FF01-609/54-25; Semrock) was acquired with the sCMOS camera using NIS-Elements imaging software (Nikon).

TIRF imaging was performed using an inverted microscope equipped with a handmaid prism-less TIR system (Axelrod, 2003 (link)). Laser sources for 488 nm and 561 nm excitation light were solid-state CW lasers (SAPPHIRE 488-20 and Compass 561-20, respectively; Coherent). Lasers were guided to the back focal plane of the objective lens (CFI Apo TIRF 60X Oil, N.A. 1.49; Nikon) through a back port of the microscope. TIR and EPI illumination were switched by tilting the incident angle of the lasers. The separated images were passed through dual-band laser split filter sets (Di01-R488/561-25×36, Di02R561-25×36, FF01-525/45-25 and FF01-609/54-25; Semrock) and captured by two EM-CCD cameras (iXon3 897; Andor) equipped with a 4× intermediate magnification lenses (VM Lens C-4×; Nikon). Cells were transferred to a cover glass (25 mm radius, 0.12-0.17 thick; Matsunami) that was fixed on a chamber (Attofluor^® Cell Chamber; Molecular Probes). The cover glass was washed by sonication in 0.1 M KOH for 30 min and in 100% ethanol for 30 min twice in advance. Cells were treated with 4 mM caffeine and 5 µM latrunculin A in DB for 15 min. Time-lapse images were obtained at 100 ms exposure for 10 min (488 nm laser power was ∼20 µW, and 561 nm laser power was ∼20 µW). Time-lapse images were smoothed with a 1 s time window to reduce shot noise.