Axioimager z2 microscope

For motility analysis in T. brucei, a sedimentation assay was conducted as previously described by Ralston et al. (2006) (link). 5×10⁶ uninduced and 6 days induced trypanosomes were incubated at 27°C in 1 ml medium in spectrophotometric cuvettes, and optical density was measured every 2 h compared to that in controls in which cells were resuspended. In L. mexicana, swimming behaviours are analysed for cells in the exponential growth phase in normal culture medium essentially as previously described (Wheeler, 2017 (link)). For cell swimming analysis, a 25.6 s video at five frames/s under darkfield illumination was captured from 5 μl of cell culture in a 250 μm deep chamber using a Zeiss Axioimager.Z2 microscope with a 10×/0.3 NA objective and a Hamamatsu ORCA-Flash4.0 camera. Particle tracks were traced automatically, and mean cell speed, mean cell velocity and cell directionality (the ratio of velocity to speed) were calculated as previously described (Beneke et al., 2019 (link)).

Leishmania cells expressing fluorescent fusion proteins were imaged live. Samples were prepared as described in [17 ]. Cells were immediately imaged with a Zeiss Axioimager.Z2 microscope with a 63× numerical aperture (NA) 1.40 oil immersion objective and a Hamamatsu ORCA-Flash4.0 camera or a 63× NA 1.4 objective lens on a DM5500 B microscope (Leica Microsystems) with a Neo sCMOS camera (Andor Technology) at the ambient temperature of 25–28°C.
For transmission electron microscopy, deflagellated cell bodies and isolated flagella were prepared with a modified version of the chemical fixation protocol described in Höög et al., [95 (link)]. Pellets of cell fractions were fixed with 2.5% glutaraldehyde in 10 mM PIPES (buffer as described above) overnight at 4°C. Pellets were washed four times for 15 min in 10 mM PIPES and overlaid with 10 mM PIPES, containing 1% osmium tetraoxide and incubated at 4°C for 1 h in darkness, then washed five times with ddH₂O for 5 min each time and stained with 500 μl of 0.5% uranyl acetate in darkness at 4°C overnight. Samples were dehydrated, embedded in resin, sectioned and on-section stained as described previously [95 (link)]. Electron micrographs were captured on a Tecnai 12 TEM (FEI) with an Ultrascan 1000 CCD camera (Gatan).

Cells from log-phase cultures were mounted on microscope slides with 0.7% LMP agarose in SC +ADE or SC-LEU +ADE, and covered with 0.17 mm glass coverslips. Our microscope system uses a Zeiss AxioImager Z2 microscope, 63X Plan Apo, 1.4NA, oil immersion objective and a Hamamatsu CCD ORCAII camera (2X2 binning and maximum analog gain). The resulting pixel size was 0.205 μm. Excitation light was provided by LED Colibri system (excitation band-pass filter): CFP 445 nm (445/25), YFP 505 nm (510/15), GFP 470 nm (474/28) and RFP 590 nm (585/35). Emission band-pass filters were as follows: CFP 47HE (480/40), YFP 46HE (535/30), GFP 38HE (525/50), and RFP 63HE (629/62). Exposure times were optimized for each fluorescent protein and ranged from 100 to 250ms. Z stacks consisted of 17 vertically separated slices with 0.4 μm spacing. The theoretical dynamic range of our system is ~3000 levels of brightness, however, in practice this will be somewhat lower.