Axioskop 2 plus microscope

All fluorescence images were captured using a Zeiss Axioskop 2plus microscope with a Hamamatsu digital camera (model C4742-95) or a Leica D6B fluorescence microscope equipped with a Leica DFC9000 GT camera and a Leica Plan Apochromat 100×/1.4 oil objective.
Microscopy of live-parasite-infected erythrocytes was performed as previously described (68 (link)). Briefly, parasites were incubated in standard culture medium with 1 μg/ml Hoechst-33342 (Invitrogen) for 15 min at 37°C prior to imaging. Infected erythrocytes (5.4 μl) were added on a glass slide and covered with a cover slip. Nuclei were stained with 1 μg/ml Hoechst-33342 (Invitrogen). Mitochondria were visualized by incubation of parasites with 20 nM MitoTracker Red 665 CMXRos (Invitrogen) for 15 min at 37°C prior to imaging. Contrast and intensities were linear adjusted for clarification and cropped images were assembled as panels using Fiji (69 (link)) and Adobe Photoshop CC 2021.

Temperature-evoked calcium dynamics in AFD were measured essentially as described previously (Takeishi et al., 2020 (link); Takeishi et al., 2016 (link); Yu et al., 2014 (link)) with the following modifications. Animals were cultivated at 20°C until the L4 stage and then shifted to the indicated temperatures. One day-old well-fed adults were immobilized in 10 mM tetramisole on an agarose pad (5% in M9 buffer) on a cover glass, and mounted under a second cover glass for imaging. The sample was transferred to a Peltier temperature control system on the microscope stage. Animals were subjected to linear temperature ramps rising at 0.05°C/s via temperature-regulated feedback using a temperature controller (Accuthermo FTC200), an H-bridge amplifier (Accuthermo FTX700D), and a thermistor (McShane TR91–170). Videos of calcium dynamics at the AFD sensory endings were captured using a Zeiss 40X air objective (NA 0.9) or a Zeiss 10X air objective (NA 0.3) on a Zeiss Axioskop2 Plus microscope, using a Hamamatsu Orca digital camera (Hamamatsu), and MetaMorph software (Molecular Devices). Data were analyzed using custom scripts in MATLAB (Mathworks) (https://github.com/wyartlab/Cantaut-Belarif-et-al.−2020) (Sternberg et al,. 2018 ; Cantaut-Belarif, 2020 (link)). T*AFD was calculated as described previously (Takeishi et al., 2016 (link)).

Images of unfixed GFP-expressing parasites were captured using a Zeiss Axioskop 2plus microscope with a Hamamatsu Digital camera (Model C4742-95, Zeiss axiovision) with 1 μg/mL DAPI (Roche) for nuclei stain. Confocal microscopy was performed using the Olympus FV1000 confocal microscope. For 3D reconstitution 20–32 z-stacks (0.3 μm step size) were collected using 488 nm laser. All confocal images were analyzed and processed in Imaris 6.2.0. Gauss filters were used with the filter width suggested by Imaris. Single images were processed in Adobe Photoshop CS4.

All fluorescence images were captured using a Zeiss Axioskop 2 Plus microscope with a Hamamatsu digital camera (model C4742-95) or a Leica D6B fluorescence microscope equipped with a Leica DFC9000 GT camera and a Leica Plan Apochromat 100×/1.4 oil objective.
Microscopy of live-parasite-infected erythrocytes was performed as previously described (93 (link)). Briefly, parasites were incubated in standard culture medium with 1 μg/mL Hoechst 33342 (Invitrogen) for 15 min at 37°C prior to imaging. A 5.4-μL portion of infected erythrocytes was added on a glass slide and covered with a coverslip. Nuclei were stained with 1 μg/mL Hoechst 33342 (Invitrogen). Microtubules were visualized by incubation of parasites in medium containing 1:1,000 TubulinTracker deep red (Thermo Fisher Scientific; dissolved in dimethyl sulfoxide [DMSO]), which labels polymerized tubulin, for 15 min at 37°C prior to imaging, as previously described (94 ). Images were processed using Fiji (91 (link)), and Adobe Photoshop CC 2021 was used for display purposes only.

Live nematodes were placed on 2% agar pads, and anaesthetised with a drop of 0.2% levamisole. Images were captured using either a Zeiss Axioskop 2 plus microscope with a Hamamatsu ORCA-ER digital camera C4742-95 and Velocity 6.3 software (Macintosh version) for image acquisition; or an ApoTome.2 Zeiss microscope with a Hamamatsu digital camera C13440 ORCA-Flash4.0 V3 and Zen software. Brightness and contrast were adjusted equally across the entire image, and where applicable applied equally to controls.