Deltavision elite microscope

For standard fluorescence microscopy, exponentially growing cells were immobilised on microscope slides covered with a thin film of 1% agarose (w/v) in water or the appropriate medium. For TIRFM, agarose pads were mounted using Gene Frames (1.7 × 2.8 cm chamber, 0.25 mm thickness, 125 µL volume) from ThermoScientific. Standard fluorescence microscopy was carried out using an Axio Zeiss Imager M1 fluorescence microscope (EC Plan-Neofluar 100x/1.30 Oil Ph3 objective) equipped with an AxioCam HRm camera and an Nikon-Ti-E microscope (Nikon Instruments, Tokyo, Japan) equipped with Hamamatsu Orca Flash 4.0 camera.
For Laurdan and TIRFM experiments, a Delta Vision Elite microscope (Applied Precision, GE Healthcare) equipped with an Insight SSI Illumination, an X4 Laser module, a CoolSnap HQ (Zhao et al., 2017 (link)) CCD camera and a temperature-controlled chamber set up at 37 ⁰C was used. Laurdan images were taken with an Olympus UplanSApo 100x/1.4 oil objective. TIRFM image series were taken using an Olympus UAPO N 100X/1.49 TIRF objective and a 561 nm laser (50 mW, 100% power). Data processing was performed with softWoRx Suite 2.0 Software.

For time-lapse movies, cells were transfected with the relevant constructs. Cells were cultured overnight, and then replated on 35 mm glass-bottomed dishes coated with one of the gelatin substrates (see above), and cultured for 1–2 h to enable cell spreading. Images were acquired by DeltaVision (RT or Elite) microscopes, using 100x/1.3 or 60x/1.42 oil objectives (Olympus), or by a DeltaVision Elite microscope equipped with total internal reflection (TIRF) optics (Applied Precision, Inc.) using 100x/1.49 TIRF or 60x/1.42 oil objectives (Olympus). The system is equipped with a temperature- and CO₂-controlled environmental box.

L. lactis was cultured until it reached an OD₆₀₀ of 0.8 and then pelleted at 8000g for
5 min and washed three times in 0.7% NaCl. The cell density was normalized
to an OD₆₀₀ of 0.4 in 0.7% NaCl, and a concentration of
5-fold MIC value of each tested antibiotic was added to the cell suspension
simultaneously with SYTO 9 and propidium iodide using the LIVE/DEAD
Baclight Bacterial Viability Kit (Invitrogen). After incubation at
room temperature for 20 min, the compounds were removed by washing
the cells with 0.7% NaCl. Finally, the cell suspensions were loaded
onto 1.5% agarose pads and analyzed with a DeltaVision Elite microscope
(Applied Precision).

Gliding assays were performed as described previously^{40 (link), 42 (link)} with some minor exceptions. Eight-well chamber slides (Thermo Fisher Scientific 154534) were coated with CSP antibodies (1:1000 in PBS). Twenty thousand salivary gland sporozoites were seeded into each well and allowed to glide for 60 min at 37 °C in 5% CO₂ in IMDM supplemented with 10% heat-inactivated human serum. Samples were fixed with 4% paraformaldehyde at 37 °C for 20 min. Primary anti-PfCSP was applied followed by goat anti-mouse Alexa 488 at (both antibodies were 1:1000 in 3% BSA). Sporozoites and trails were viewed on a Deltavision Elite microscope (Applied Precision) using an Olympus 163x/1.42 PlanApoN objective equipped with a Coolsnap HQ2 CCD camera. A total range of 220 (NF54) to 840 (ΔPOFUT2) sporozoites were counted for each condition across two independent experiments.

Yeast cells were grown and processed for fluorescence microscopy as described previously [72 (link)]. Fluorophores were cyan fluorescent protein (CFP, clone W7) [73 (link)], yellow fluorescent protein (YFP, clone 10C) [74 (link)] and red fluorescent protein (RFP, clone yEmRFP; or mCherry) [75 (link)]. Fluorophores were visualized on a Deltavision Elite microscope (Applied Precision, Inc) equipped with a 100x objective lens (Olympus U-PLAN S-APO, NA 1.4), a cooled Evolve 512 EMCCD camera (Photometrics, Japan), and an Insight solid-state illumination source (Applied Precision, Inc). Pictures were processed with Volocity software (PerkinElmer). Images were acquired using softWoRx (Applied Precision, Inc) software.
Spontaneous Rad52-YFP foci from mid-log growing cells carrying plasmid pWJ1344 were visualized and counted by fluorescence microscopy as described in [76 (link)].