Planapo n 60 1.42 na oil objective

To prepare imaging slides, coverslips of thickness No. 1.5H (0.170 mm ± 0.005 mm) were flamed and smeared with 0.1% Polyethylenimine (PEI) solution. Purified ookinetes or salivary gland sporozoites were fixed with 2%PFA in 1×PBS suspension and allowed to settle onto PEI-treated coverslips for 20 minutes and then probed with indicated antibodies (13.1 mouse monoclonal antibody for ookinetes and CSP antibody for sporozoites) by IFA, stained with DAPI, rinsed in water and mounted onto Vectashield on glass slides before sealing with nail varnish. Super-resolution images were acquired using a Deltavision OMX 3D-SIM System V3 BLAZE from Applied Precision (a GE Healthcare company) equipped with 3 sCMOS cameras, 405, 488, 592.5 nm diode laser illumination, an Olympus Plan Apo N 60 × 1.42NA oil objective, and standard excitation and emission filter sets. Imaging of each channel was done sequentially using three angles and five phase shifts of the illumination pattern as described previously49 (link). The refractive index of the immersion oil (Cargille) was adjusted to 1.516 to minimize spherical aberrations. Sections were acquired at 0.125 μm z steps. Raw OMX data was reconstructed and channel registered in SoftWoRx software version 6.1.3 (Applied Precision, a GE Healthcare company).

Differentiated SH-SY5Y cells (6500 cells/well) and DIV6-hippocampal neurons (50000 cells/well) were co-transfected with CMV-eGFP-NMIIA (Addgene, cat# 11347) and NMIIA-mApple (a kind gift from Dr John Hammer) using 1 µg:1 µg of each construct/well and Lipofectamine 3000 following the manufacturer’s instructions. Two days later, in the case of SH-SY5Y cells, and four days later (at DIV10) in the case of primary hippocampal neurons, the cells were fixed. Transfected cells were then imaged using an Olympus SpinSR10 spinning disk confocal super‐resolution microscope (Olympus, Tokyo, Japan) equipped with an PlanAPON 60 ×/1.42 NA oil objective (Olympus), a CSU-W1 SoRa-Unit (Yokogawa, Tokyo, Japan) with 3.2x magnification and ORCA‐Flash 4.0 V3 Digital CMOS Camera (Hamamatsu, Hamamatsu City, Japan).