Ixon 897 emccd camera

Cells were imaged with a Nikon Ti-E microscope equipped with a motorized TIRF Illuminator unit, a 60x TIRF objective (60x Plan Apo, Oil DIC N2, NA =1.49, WD = 120 um) and Perfect Focus System. Images were acquired with an Andor iXon 897 EMCCD camera and the Nikon NIS elements software. mNeonGreen was imaged using the 488 nm laser line and calcein red-orange was imaged using the 561 nm laser line. A quad split dichroic mirror (405 nm, 488 nm, 561 nm, 640 nm) was used in combination with dual band pass emission filter (515 to 545 nm, 600 to 650 nm). To achieve a larger field of view a 3 x 3 tile scans was acquired with 15% overlap stitching on the GFP channel. Time lapse images were taken every 10 s.

Cells were seeded on glass bottom gridded dishes (MatTek) and live-cell imaging was performed as described above. Cells were fixed on stage by adding 3.7% formaldehyde at 1:1 ratio to culture medium and a differential interference contrast (DIC) image was captured with a × 10 0.3 NA lens to mark the position of the cells imaged on the grid. After appropriate permeabilisation and immunolabelling for ATG13, coverslips were mounted into custom imaging rings and immersed in 500 μl STORM imaging buffer, transferred to a Nikon N-SIM/N-STORM dual super-resolution microscope (Nikon Ti-E microscope with SIM/STORM adaptations, Andor iXon 897 EM-CCD camera and Nikon 1.49 NA oil immersion objective, with the imaging system controlled using Nikon Elements software) and cells were relocated and imaged first using SIM and thenSTORM. Raw 3D-SIM images were acquired (15 images representing 5 phases and 3 rotations at each focal plane) typically using 100 msec exposure, 5.1 conversion gain and 150 electron microscopy gain and reconstructed into super resolved images using default parameters in the Nikon software. mCherry was excited using a 561 nm laser with emission collected at 607/36; Alexa Fluor 647 was excited using a 643 nm laser with the emission collected at 692/40.

TIRF microscopy images of HeLa and HEK293T cells were acquired with a Nikon Ti-E microscope equipped with a motorized TIRF Illuminator unit, a 60× TIRF objective (60× Plan Apo, Oil DIC N2, NA=1.49, WD=120 μm), Perfect Focus System, with an Andor iXon 897 EMCCD camera and the Nikon NIS elements software. CFPs were imaged using the 440 nm laser line in combination with a tri split dichroic mirror (440, 488 and 561 nm). GFPs and YFPs were imaged using a 488 nm laser line in combination with a quad split dichroic mirror (405, 488, 561 and 640 nm) and a dual band pass emission filter (515–545 nm and 600–650 nm). RFPs were imaged using a 561 nm laser line in combination with a quad split dichroic mirror (405, 488, 561 and 640 nm) and a dual-band pass emission filter (515–545 nm and 600–650 nm). Photo activation was achieved with a 440 nm laser line, intensity set to 20%, for 1 s repeated for every imaged frame.
TIRF microscopy images of SUM159 cells were acquired at a Nikon Eclipse Ti2 microscope, equipped with a Tokai Hit STX stage top incubator (set to 37°C and 5% CO₂ for live imaging), Apo TIRF 60× NA 1.49 oil immersion objective, Hamamatsu ORCA-Flash 4.0 camera, Agilent laser unit (405, 488, 561 and 647 nm), and NIS-Elements AR software.