Plan apochromat 63x 1.40 oil m27 objective

Subcellular localization of αSyn-GFP in living cells was assessed via confocal microscopy using a ZEISS LSM800 Airyscan microscope (Fig 1C), equipped with a Plan-Apochromat 63x/1.40 Oil M27 objective, and ZEISS ZEN software control. Micrographs in Figs 2B and 3E were recorded with a Leica SP5 confocal laser scanning microscope, equipped with a Leica HCX PL Apo 63x NA 1.4 oil immersion objective. Cells were counterstained with PI to visualize dead cells and subsequently immobilized on agar slides. Micrographs were processed with the open-source software Fiji [105 (link)]. Gaussian filtering (σ = 1) was applied to reduce image noise, followed by background subtraction (rolling ball radius = 50 pixels). Pictures within an experiment were captured and processed using the same settings.

For fluorescence-in-situ-hybridizations (FISH), 50 µl of fixed biofilm sample was diluted in 1 ml 1 × PBS, filtered on a 25 mm polycarbonate filter (0.2 µm pore size) and embedded in 0.2% w/v Agarose. FISH was done using double-labelling-of-oligonucleotide-probes (DOPE; Stoecker, et al.^{26 (link)}) for Archaea (ARCH915; Stahl and Amann²⁷ ) and Desulfobacteraceae (DSS658; Mußmann, et al.^{28 (link)}) synthesized by biomers.net GmbH (Ulm/Donau, Germany). The antisense probe NON338^{29 (link)} was used to test for unspecific staining for the given formamide concentrations. The probes were labelled at the 5′ and 3′ end with Cyanine 3 (ARCH915) and 6-FAM (DSS658, NON338). Hybridizations were done in accordance to published work^{30 (link)} with 3 h hybridization with DSS658 (50% formamide) followed by ARCH915 (0% formamide). The NON338 probe was incubated for 3 h (0% formamide). After DOPE FISH, the samples were counterstained with DAPI as described by Glöckner et al.^{31 (link)}. Imaging was done with a confocal laser scanning microscope (Axio Observer LSM800, Carl Zeiss Microscopy GmbH, Jena, Germany) using a Plan-Apochromat 63x/1.40 Oil M27 objective. Emission and detection wavelengths were 561 and 535–700 nm for Cy3, 488 and 450–545 nm for 6-FAM, and 405 and 400–600 nm for DAPI.