Hard and soft plastic resin embedding for single-cell element uptake investigations of marine-snow-associated microorganisms using nano-scale secondary ion mass spectrometry: NanoSIMS of sectioned marine snow samples

Marine snow aggregates are microhabitats for diverse microbial communities with various active metabolic pathways. Rapid recycling and symbiotic transfer of nutrients within aggregates poses a significant challenge for accurately assessing aggregate-associated turnover rates. Although single-cell uptake measurements are well-established for free-living microorganisms, suitable methods for cells embedded in marine snow are currently lacking. Comparable cell-specific measurements within sinking pelagic aggregates would have the potential to address core questions regarding aggregate-associated fluxes. However, the capacity to perform microscale studies is limited by the difficulty of sampling and preserving the fragile aggregate structure. Furthermore, the application of nano-scale secondary ion mass spectrometry (NanoSIMS) to aggregates is complicated
by technical requirements related to vacuum and ablation resistance. Here, we present a NanoSIMSoptimized method for fixation, embedding, and sectioning of marine snow. Stable isotope labeling of laboratory-generated aggregates enabled visualization of label incorporation into prokaryotic and eukaryotic cells embedded in the aggregate structure. The current method is also amenable to various staining procedures, including transparent exopolymer particles, Coomassie stainable particles, nucleic acids, and eukaryotic cytoplasm. We demonstrate the potential for using structural stains to generate three-dimensional (3D) models of marine snow and present a simplified calculation of porosity and fractal dimension. This multipurpose method enables combined investigations of 3D aggregate structure, spatial microbial distribution, and
single-cell activity within individual aggregates and provides new possibilities for future studies on microbial interactions and elemental uptake within marine snow.