English: Environmental high-content fluorescence microscopy (e-HCFM): automated, 3D, and multichannel imaging for aquatic micro-eukaryotes.
(a) e-HCFM workflow applied to Tara Oceans samples: (1) 72 nano-plankton (size range 5–20 μm) samples collected during the Tara Oceans expedition (Pesant et al., 2015) were fixed in paraformaldehyde-glutaraldehyde buffer onboard and kept at 4°C for up to several years; (2) Samples were mounted in optical multiwell plates. Then, a 4-steps preparation allowed to stain all eukaryotic cells; (3) A commercial confocal laser scanning microscope was used to automatically image samples (40X NA1.1 water lens; 5 channels) generating 2.5 Tb of raw data (acquisition details in Figure 1—source data 1); (4) In total, 336,655 objects were processed for individual extraction of 480 descriptors (3D biovolumes, intensity distribution, shape descriptors and texture features, details in Figure 1—source data 2), and the reconstruction of various images for visual inspection (c); (5) A training set based on 18,103 manually curated images (5.4% of the dataset) classified into 155 categories, was used for automated recognition (Random Forest). (b) Examples of e-HCFM 3D-images and movies from a wide phylogenetic diversity of planktonic eukaryotes (see also Figure 1—figure supplements 1 and 2). Left panel: a chain of diatoms (Asterionellopsis sp., Heterokonta) (Figure 1—video 1); right panel, top left to bottom right: radiolarian (Rhizaria), ciliate (Alveolata), amoeba (Amoebozoa), choanoflagellate (Opisthokonta), dinoflagellate (Alveolata), coccolithophore (Haptophyta). Key cellular features are labelled with various dyes: DNA/nuclei (blue, Hoechst33342); (intra)cellular membranes (green, DiOC6(3)); cell covers and extensions (cyan, PLL-AF546, a home-made conjugation between α-poly-L-lysine (PLL) and Alexa Fluor 546 (AF546)); chloroplasts (red, chlorophyll autofluorescence). Scale bar 5 µm. (c) The confocal microscope is automated for acquiring multicolor Z-stacks over a mosaic of positions for each sample.