Teneo sem

A short mooring line (~50 m long) was deployed at 42° 01′N–4° 48′E (depth of 2400 m). It was equipped with a Technicap PPS-3 sediment trap (collecting area of 0.125 m², aspect ratio of 2.5, and 12 collecting cups) at 30 m above the seabed. The trap samples were collected with sampling interval between 15 and 23 days. Prior to deployment, the sampling bottles were filled with 0.45 µm filtered seawater containing sodium borate-buffered formalin to yield a final concentration of 5% formalin to prevent in situ microbial decomposition. Upon recovery, samples were stored in the dark at 4 °C^{76 (link)}. 1 mL aliquots were filtered onto the center of 0.4 µm polycarbonate filter using a filtering funnel of 6 mm aperture, and carefully rinsed with DIW water, then dehydrated in increasing series of ethanol 30%, 50%, 70%, 80%, 90%, and 100% during 10 mn for each step. The samples were completely dried overnight, mounted on aluminum stubs with double sticky carbon tabs, and sputter-coated with gold for 10 min. The samples from each of the three available trap samples were analyzed with a FEI Teneo SEM.

Whole infected RBCs were fixed and imaged as previously described [25 (link)]. Scanning electron microscopy (SEM) imaging of the cytoplasmic surface of the host RBC membranes was performed on sheared infected RBC membranes. The membranes were prepared as previously described [25 (link)]. Briefly, glass coverslips were cleaned with acetone and 50% methanol and functionalized with 3-aminopropyl triethoxysilane (APTES), bis-sulfosuccimidyl suberate, and erythroagglutinating phytohemagglutinin (PHAE). Mature infected-RBCs were immobilized on the functionalized glass slides and sheared by forceful application of a hypotonic buffer (5 mM Na₂HPO₄/NaH₂PO₄, 10 mM NaCl, pH 8). For CS2 and ΔPTP7 cells, coverslips were coated with gold at 25 mA for 40 s and 75 s using a Dynavac sputter coating instrument to thicknesses of ~0.2 nm and ~0.4 nm for the sheared and whole cells, respectively. Otherwise, whole cells were gold-coated with a Safematic CCU-010 sputter coater to a thickness of 5 nm. Images were acquired with the T1 (A+B, for CS2 and ΔPTP7 whole cells) or the Everhart-Thornley detector in ‘Optiplan’ mode (for whole cells and sheared membranes) of an FEI Teneo SEM using a working distance of 5 mm, a beam current level of 50 pA, and 2 kV accelerating voltage.

Decidual tissue with diameter of 1 mm from normal pregnant mice in the NP group and aborted mice of the A30E group were fixed in 2.5% glutaraldehyde for 2 h, then fixed in 1% osmic acid at 4 °C for 1 h. After washed with 0.1 M PBS, tissue samples were in turn dehydrated and with 50% ethanol, 70% ethanol, 90% ethanol, 100% ethanol (3 times), then ethanol:isoamyl acetate (1:1), and 100% isoamyl acetate. Samples were dried on a Samdri®-795 ‍Critical point drier (Tousimis, U.S.A.) and sprayed with 10 nm metal conductive thin layer on an EMS 550 Ion beam sputtering coating instrument (EMS, U.S.A.). Samples were then observed and imaged under scanning electron microscope Teneo SEM (FEI, U.S.A).