Intravital imaging was performed in 5- to 8-week-old female UBC-GFP C57BL/6 mice, Lys-GFP C57BL/6 mice, Flk1-GFP mice, vascular endothelial-cadherin-GFP mice, WT C57BL/6 mice, and WT Balb/c mice infected with Pb-mCherryHsp70 parasites using synchronous stages (that is, schizonts, merozoites, trophozoites, and gametocytes). Three parasite doses were injected for all experiments involving synchronous infections: an initial inoculum of 105, 106, or 6 × 106. A mixture of anesthetics comprising ketamine (125 mg/kg; Ketasol, Graeub) and xylazine (12.5 mg/kg; Xylasol, Graeub), was prepared and diluted in 1× PBS (1:2:5). Mice were injected intraperitoneally with 100 ml per 20 g of body weight, of the mixed anesthetic, and injections were repeated periodically during imaging. In the case of solid organs, the surgical procedure involved a small incision to expose the spleen, kidney, liver, or adipose tissue. The exposed tissues were glued to a microscope cover glass (VWR 24, 50 mm, no.1) and imaged with a Zeiss LSM5 live scanning module, a Leica SP8-STED confocal microscope, or a Andromeda confocal Spinning Disc microscope with Yanus laser scan head, from Till Photonics. In each microscope, imaging was performed using a Plan Apochromat 63× 1.4 numerical aperture (NA) DIC M27 oil immersion objective (LSM5), an HC PL APO CS2 63× 1.4 NA or 100× oil immersion objective (SP8-STED), or a 60× 1.4 NA oil immersion objective from Olympus. All microscopes were equipped with a temperature chamber to enable controlling the mice’s body temperature following anesthesia during image acquisition. In the Zeiss LSM5 confocal microscope, imaging was performed with 0.1 mW from a 489-nm light from a diode laser to excite GFP, whereas 2 mW from a 561-nm diode pumped solid-state laser was used to excite mCherry. Simultaneous, rather than sequential, excitation was used to ensure high-speed image acquisition. In the Leica SP8-STED microscope, a white laser was used, and wavelengths were defined for the respective spectra of GFP and mCherry. As above, 512 × 512 fields of view were acquired, at speed 4, zoom 10 to 40, and pinhole 1.0. For fast image acquisition, dual fluorescence was simultaneously acquired using an 8000-Hz resonant scanner with 30 times frame averaging. In the Andromeda confocal spinning disc system, GFP was excited with a 488-nm laser, and mCherry was excited with a 561-nm laser. Between 50 and 100 different fields of view were selected in each mouse organ, and images were acquired every 500 ms for 5 min. Experiments were done in triplicate mice for each synchronous parasite stage, each day after infection (that is, 2 hours, days 1 to 7), and the different initial infectious inocula (that is, 105, 106, or 6 × 106). Line scans were positioned at various depths of the vessel, and video-rate imaging was used to record blood and parasite flow in P. berghei–infected mice. The recorded movies were assembled and processed using Fiji software (http://fiji.sc/Fiji).

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