Plan neofluar 10x 0.3 objective

To assess gliding motility, 5,000 sporozoites were resuspended in 1x PBS containing 5% FCS and 10 μg/mL of hmAb in a final volume of 20 μL. The resulting suspension was transferred to an 18-well slide (iBidi) and centrifuged at 400 g for 3 min at 4°C. The slide was then allowed to equilibrate at 37°C, 5% CO₂ for 3 min in the incubation chamber (Incubation System S, Zeiss) of an inverted epifluorescence wide-field microscope (AxioObserver Z.1, Zeiss) equipped with a LED illumination system (Colibri2, Zeiss), a CCD camera (AxioCam MR, Zeiss) and controlled by the AxioVision software (version 4.8.2.0, Zeiss). Time-lapse movies were then recorded for 2–4 min at a rate of one image per second with an EC “Plan-Neofluar” 10x/0.3 objective (Zeiss) using a 470 nm LED and a matching filter cube (43HE, Zeiss) to excite and detect GFP and thus visualize sporozoites. Average sporozoite velocity over the first 2 min of the acquisition was determined using the MTrack2 plug-in from Fiji.^{38 (link)}

After mouse cardiac perfusion with PBS supplemented with 5 mM of EDTA, mouse hearts were harvested and fixed in 4% paraformaldehyde (PFA) for 24 h at 4 °C, incubated 24 h in PBS supplemented with 30% sucrose, embedded in OCT and cryopreserved at −70 °C.
Cryocut cross-sections (5 µm) of aortic roots were evaluated for conventional hematoxylin-eosin (HE) staining, 0.1% sirius red to detect collagen or 0.5% ORO to detect neutral lipids. Images were captured with a Zeiss Axiophot microscope with a Plan-NEOFLUAR 10x/0.3 objective (Zeiss, Oberkochen, Germany) and a DP70 camera (Olympus, Madrid, Spain). Polarized images were obtained using a Mirax digital slide scanner (3DHistech, Budapest, Hungary). To avoid specific biases due to potential differences in lesion shape, cross sections of the entire lesion were analyzed and averaged [17 (link)].
To obtain the aortas for the analysis, after fixing in PFA overnight at 4 °C, the aortas were whole-mount stained with 0.2% ORO in methanol, opened longitudinally and pinned to black wax to expose the entire luminal surface. Images were acquired using a Leica MZ6 SZX10 stereomicroscope (Leica Microsystems, Wetzlar, Germany) coupled to a Leica DFC300 digital color camera (Leica Microsystems). The planimetric area of atherosclerotic plaques was measured in pixels using ImageJ.

Black carbon particles in the different brain regions were detected via a specific and label-free technique based on the nonincandescence-related white light generation of the particles under femtosecond-pulsed illumination, as previously described by our research group.^{10 (link),12 (link),13 (link)} Images were taken with a confocal microscope (Carl Zeiss) equipped with a 2-photon femtosecond-pulsed laser (810 nm, 150 fs, 80 MHz) (Spectra-Physics) using a Plan-Neofluar 10x/0.3 objective (Carl Zeiss). Two-photon–induced white light emission of carbon particles was acquired in the nondescanned mode after spectral separation and emission filtering using 400-410 nm and 450-650 nm band-pass filters. The resulting tile scans of 13.35 μm × 13.35 μm were recorded with a 1.66 μm pixel size and 4.10 μs pixel dwell time.