Softworx 5

Glass coverslips were incubated with 0.1 mg/ml lectin from Phaseolus vulgar (PHAE) (Sigma-Aldrich) for 30 min at 37°C in a humidity chamber. After 3 washes in PBS, infected RBCs (3% hematocrit) were incubated for 10 min on the coverslip. Nuclei were stained using 2 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) for 10 min. Slides were mounted in p-phenylenediamine antifade. Images were taken on a Delta Vision elite restorative widefield deconvolution imaging system (GE Healthcare) using a 100× UPLS Apo objective (1.4 numeric aperture; Olympus) lens under oil immersion. The following emission/excitation filter sets were used: DAPI, excitation at 390/18 and emission at 435/48; fluorescein isothiocyanate, excitation at 475/28 and emission at 523/26. Images were deconvoluted using Softworx 5.0 (GE Healthcare) and analyzed using ImageJ (NIH).

For live cell imaging experiments, cells were seeded onto glass-bottom dishes (627870, Greiner Bio One) a minimum of 16 h prior imaging. Treatments were carried out in phenol red-free DMEM buffered with 20 mM Hepes pH 7.4, and imaging was carried out by widefield deconvolution microscopy (Deltavision Elite, GE Healthcare) in a heated environment chamber. Images were acquired and deconvolved using softWoRx 5.0 (Applied Precision, GE Healthcare).
For long-term starvation treatments, cells were maintained in a humidified environmental chamber with 5% CO₂ and imaged by bright-field (Eclipse TiE, Nikon Instruments). Images were acquired and analysed utilising NIS-Elements (Nikon Instruments).

For wide‐field imaging, living SCs were imaged using a DeltaVision Elite wide‐field fluorescence deconvolution microscope (GE Healthcare Life Sciences) equipped with both a 100×, NA 1.4 UPlanSApo oil objective (Olympus) and a 60×, NA 1.42 UPlanSApo oil objective (Olympus), as well as a CoolSNAP HQ2 CCD camera (Photometrics^®) and a seven‐colour illumination system (SPECTRA light engine^®, Lumencor). A 1.6× auxiliary magnification lens and objective immersion oil with RI 1.514 (Cargille Labs) were used throughout. The microscope room was temperature‐controlled at ~ 18°C. The images acquired were typically Z‐stacks spanning 8–12 μm depth with a z‐distance of 0.2 μm. Images were subsequently deconvolved using the Resolve 3D‐constrained iterative deconvolution algorithm within the softWoRx 5.5 software package (GE Healthcare Life Sciences).