Intravital and ex vivo imaging

For intravital imaging, surgeries were separately performed in the brain; the lungs and heart; the liver; the pancreas, spleen and kidney; the adipose tissues and lymph nodes, as described in De Niz et al. (2019a, 2019b, 2019c, 2020). Briefly, mice were anaesthetized with a mixture of ketamine (120 mg/kg) and xylazine (16 mg/kg) injected intraperitonially. After checking for reflex responses and ensuring none occurred, mice were intraocularly injected with Hoechst 33342 (stock diluted in dH₂O at 100 mg/ml; injection of 40 μg/kg mouse), 70 kDa FITC-Dextran (stock diluted in 1x PBS at stock concentration of 100 mg/ml; injection of 500 mg/kg), and vascular markers of interest conjugated to A647 (CD31 (Biolegend, used at 20 μg), Ephrin B2 (R&D systems, used at 20 μg), Eph-B4 (R&D systems, used at 20 μg), or VEGFR3 (R&D systems, used at 20 μg). A temporary glass window (Merk rectangular coverglass, 100 mm x 60 mm or circular coverglass (12 mm)) was implanted in each organ, and secured either surgically, with surgical glue, or via a vacuum, in order to enable visualization of the organ surface.

For intravital microscopy, all imaging relative to parasite quantifications, vascular density and vascular leakage was done in a Zeiss Cell Observer SD (spinning disc) confocal microscope (Carl Zeiss Microimaging, equipped with a Yokogawa CSU-X1 confocal scanner, an Evolve 512 EMCCD camera and a Hamamatsu ORCA-flash 4.0 VS camera) or in a 3i Marianas SDC (spinning disc confocal) microscopy (Intelligent Imaging Innovations, equipped with a Yokogawa CSU-X1 confocal scanner and a Photometrics Evolve 512 EMCCD camera). Laser units 405, 488, 561 and 640 were used to image Hoechst in nuclei, extravascular and intravascular FITC-Dextran, TdTomato in T. brucei, and CD31, respectively. The objective used to image vascular density, vascular leakage, and proportion of intravascular and extravascular parasites was a 40x LD C-Apochoromat corrected, water immersion objective with 1.1 NA and 0.62 WD. The objective used to classify T. brucei movement phenotypes was a 100x plan-apochromat, oil immersion objective with 1.4 NA and 0.17 WD. Between 20 and 100 images were obtained in any one time lapse, with an acquisition rate of 20 frames per second. For vascular density measurements and parasite quantification, in order to gain access to the full organ, we performed ex vivo imaging from different organ regions. For this, we performed z stacks consisting of 16 stacks covering up to 200 μm of tissue depth. For all acquisitions, the software used was ZEN blue edition v.2.6 (for the Zeiss Cell Observed SD) allowing export of images in .czi format, and 3i Slidebook reader v.6.0.22 (for the 3i Marianas SD), allowing export of images in TIFF format.