Softworx 7

Super‐resolved fluorescence images were reconstructed with the corresponding recorded optical transfer function (OTF) in the softWoRx 7.0.0 software (GE Healthcare, Amersham, UK) at a Wiener filter setting of 0.006.

Transfected cells were grown to confluency on glass coverslips before staining with 100 nM MitoTracker Green for 30 min at 37 °C in a complete growth medium. Coverslips were washed in HBSS and incubated in a complete growth medium in Attofluor Cell Chambers (Invitrogen) for live imaging using super-resolution 3D structured illumination microscopy (SR-3D SIM) on an OMX v4 microscope (GE Healthcare) at 37 °C, using 488 and 568 nm laser lines and a 60x NA PLAPON oil objective. Image stacks were acquired with an optical spacing of 125 nm. Super-resolution images were reconstructed and registered with softwoRx 7.0 (GE Healthcare). The necessary optical transfer functions and channel registrations were acquired shortly before.

Super-resolution three-dimensional structured illumination microscopy (3D SIM) was performed on the DeltaVision OMX-SR system (GE Healthcare) equipped with a ×60/1.42 NA PlanApo oil immersion objective (Olympus), sCMOS cameras, and 405, 488, 568, and 640 nm lasers, and 1.518 or 1.520 refractive index immersion oil. To image RON3 localization on late-stage parasites, synchronized mNeonGreen-tagged RON3 schizonts were labeled with Sir-DNA (Spirochrome) nucleus dye. The 488 nm laser was used to excite mNeonGreen and the 640 nm laser was used to excite Sir-DNA. To image RON3 localization on samples fixed upon invasion assay, the 405 nm laser was used to excite DAPI, the 488 nm to excite mNeonGreen-tagged RON3, and the 640 nm to excite the wheat germ agglutinin membrane label. Structured Illumination image stacks were constructed from 15 raw images per plane (five phases, three angles) per color channel and a z-step size of 125 nm. Images for widefield deconvolution were recorded under standard widefield excitation with a single recorded image per color channel per z-stack with a z-step size of 375 nm. Super-resolution reconstruction or widefield deconvolution and color channel alignment were performed with softWoRx 7.0 (GE Healthcare).

Super-resolution three-dimensional structured illumination microscopy (3D SIM) was performed on the DeltaVision OMX-SR system (GE Healthcare) equipped with a 60×/1.42 N.A. PlanApo oil immersion objective (Olympus), sCMOS cameras, and 488 and 568 nm lasers, and 1.516 refractive index immersion oil. 3D SIM image stacks were acquired consisting of 15 raw images per plane (5 phases, 3 angles) per color channel and a z-step size of 125 nm. Super-resolution reconstruction and color channel alignment were performed with softWoRx 7.0 (GE Healthcare).