Elyra super resolution microscopy

Trypanosomes were harvested by centrifugation and fixed with 4% paraformaldehyde in Phosphate Buffered Saline (PBS, supplemented with 250 mM sucrose in case of RNAi experiments) for 15 min at 4°C. After two washes, the fixed cells were resuspended in PBS and immobilized on poly-L-lysine (Sigma) coated wells, further permeabilized with PBS containing 1% Triton X-100 and blocked with blocking buffer (PBS containing 3% BSA and 0.25% Tween 20). To study the subcellular localization, α-TbAldolase (1:500 dilution in blocking buffer) was used as glycosomal marker, while Rabbit Alexa Fluor 594 (Thermo Fischer Scientific) at 1:1000 in blocking buffer was used as secondary antibody. The Nuclear and kinetoplast DNA were stained with DAPI. Stained cells were layered with Mowiol (Sigma) antifade-medium and covered with coverslips. After an overnight setting time that allows the polymerization of Mowiol, the images were captured using Zeiss ELYRA Super Resolution Microscopy and analyzed using Zen 3.6 (blue edition) (Carl Zeiss Microscopy GmbH).

Wide-field microscopy was performed using an AxioImager 2 (Zeiss) employing either a 63× or 100× objective. Confocal microscopy was performed with an LSM 880 (Zeiss). SIM was performed using an Elyra superresolution microscopy (Zeiss), using five rotations of the illumination pattern. Image processing (reconstruction and channel alignment) was conducted using Zen Black edition (Zeiss), and 3D rendering was performed using Volocity (Perkin-Elmer).