Elyra structured illumination microscope

Samples were fixed shipboard using freshly prepared paraformaldehyde (2 vol% in 3× Phosphate Buffer Solution (PBS), EMS15713) at 4 °C overnight, rinsed twice using 3× PBS, and stored in ethanol (50% in 1× PBS) at −20 °C until processing. Small pieces (<1 cm³) of the mineral sample NA091.008 were gently crushed in a sterile agate mortar and pestle in a freshly prepared, filter sterilized 80% ethanol – 1× PBS solution. About 500 μl of the resulting mixture was sonicated three times in 15 second bursts on a Branson Sonifier W-150 ultrasonic cell disruptor (level 3) on ice with a sterile remote-tapered microtip probe inserted into the liquid. Cells were separated from the mineral matrix using an adapted protocol of density separation using Percoll (Sigma P4937)^{7 (link)}. The density-separated cells were filtered on 25 mm polycarbonate filters with a pore size of 0.22 μm (Millipore GTTP2500), and rinsed using 1× PBS. Fluorescence in situ hybridizations were carried out as described previously^{7 (link)} using a 1:1 mixture of an ANME-1 targeted probe (ANME-1-350^{9 (link)} labelled with Cy3) and the general bacterial probe mix EUB338 I-III (https://probebase.csb.univie.ac.at/), labelled with Alexa-488 in a 35% formamide solution (VWR EM-FX0420-8). Hybridized samples were imaged using a ×100 objective using a Zeiss Elyra structured illumination microscope with the Zen Black software.