Zyla 4.2 plus camera

All FRET ratio‐imaging experiments were performed on an epifluorescence Eclipse Ti inverted fluorescence microscope (Nikon) with a PlanApo air 20× (NA 0.75) objective controlled by NIS‐Elements (Nikon). Laser‐based autofocus was used throughout the experiments. Image acquisition was performed with an Andor Zyla 4.2 Plus camera at a 16‐bit depth. Donor, FRET, and red channel images (to visualize an Alexa 546‐dextran that indicates GF exposure) were acquired sequentially using filter wheels. The following excitation, dichroic mirrors, and emission filters (Chroma) were used: donor channel: 430/24×, Q465LP, 480/40 m; FRET channel: 430/24×, Q465LP, 535/30 m; and red channel: ET550/15, 89,000 bs, 605/50 m (for dextran imaging). Standard exposure settings were used throughout the experiments. 440‐nm (donor and FRET channel excitation) and 565‐nm (red dextran) LED lamps were used as light sources (Lumencor Spectra X light engine), with 3% (440 nm) and 5% (565 nm) of lamp power. Acquisition times were 30 ms for donor channel and 30 ms for FRET at binning 2 × 2 and 100 ms 8 × 8 binning for the red channel. Cells were imaged in DMEM with 1,000 mg/ml glucose, and penicillin/streptomycin, at 37°C. The microfluidic device was mounted on the microscope stage and was connected by the tubing to a CellASIC ONIX (Merck Millipore) pump.

The fixed mouse intestinal organoid, the live zebrafish embryo, the macrophages infected with Mycobacterium smegmatis bacteria and the fixed actin-stained COS-7 cells were imaged using a 20x and 100x (1.3 NA, oil immersion) objective. The 100 nm bead sample and the fixed bovine pulmonary artery endothelial cells were imaged with a 100x objective (1.3 NA, oil immersion). For the image acquisition an Andor Zyla 4.2 Plus camera (pixel format 1048 × 1048) and an Andor iXon3 camera (pixel format 1024 × 1024) were used. We provide example data sets of the fixed 100 nm bead sample, fixed bovine pulmonary artery endothelial cell sample and the live zebrafish sample on Zenodo⁴³ including the raw data and the reconstructed data with corresponding information about the used parameter setting during image acquisition and image reconstruction.

Plasmid ptfLC3 was a gift from Tamotsu Yoshimori (Obtained from Addgene, plasmid # 21074)^{66 (link)}. Transfections of cell monolayers were done with the Lipofectamine Plus reagent (Invitrogen), according to the manufacturer’s instructions. Transfected cells were incubated at 37 °C for 18 to 24 h, unless otherwise stated. For immunofluorescence studies, the cells were permeabilized by incubation with 0.1% Triton X-100/PBS for 10 min at room temperature followed by incubation with 5% BSA/PBS for 10 min. The primary antibodies indicated for any particular experiment were added to cell monolayers in 5% FBS/PBS and incubated for 2 h at rt. After washing the monolayers three times with 5% FBS/PBS, cells were incubated for 1 h with secondary antibodies. Images were alternatively obtained with an Olympus DP-71 digital camera mounted on an Olympus BX51 fluorescence microscope, or with an Olympus IX70 equipped with a TillPhotonics camera. Confocal images were acquired either with an Olympus FV1000 confocal microscope or with an Andor Dragonfly spinning disk confocal system mounted on a Nikon TiE microscope equipped with a Zyla 4.2 PLUS camera (Andor). Colocalization was quantified as Pearson’s coefficient using JACoP, ImageJ plugin^{67 (link)}.