Assigning Function to Phylogeny: FISH-nanoSIMS.

Catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) is an imaging method used to identify microorganisms in environmental samples based on their phylogeny. CARD-FISH can be combined with nano-scale secondary ion mass spectrometry (nanoSIMS) to directly link the cell identity to their activity, measured as the incorporation of stable isotopes into hybridized cells after stable isotope probing. In environmental microbiology, a combination of these methods has been used to determine the identity and growth of uncultured microorganisms, and to explore the factors controlling their activity. Additionally, FISH-nanoSIMS has been widely used to directly visualize microbial interactions in situ. Here, we describe a step-by-step protocol for a combination of CARD-FISH, laser marking, and nanoSIMS analysis on samples from aquatic environments.