Generally considering the widespread distribution and functional importance of microorganisms in biogeochemical processes, single-cell methods to detect their metabolic activities in naturally complex environments are essential. Recent methodological advancements in secondary ion mass spectrometry (SIMS)-based imaging techniques have revealed novel insights into metabolically active cells and their identification. The nano-scale secondary ion mass spectrometry (NanoSIMS) represents the most advanced generation of ion microprobe imaging technique, which possesses well-focused primary ion beam, high levels of sensitivity, ion transmission, mass resolution and spatial resolution (< 50 nm), combining high-resolution microscopy with isotopic analysis. NanoSIMS has been widely used in material science, geology, life science and mineralogy, and recently expanded to be a novel analytical tool in environmental microbiology.
The basic principle of NanoSIMS is that the primary ions produced by the ion source (Cs⁺ or O^-) are accelerated under ultra-high vacuum to an energy with a few kiloelectronvolts (KeV), then are focused onto the chosen working areas of the sample surface. The bombardment could sputter away a thin layer of secondary ions reflecting the molecular and isotopic compositions of sample surface. These ion particles are directed into a mass analyzer, and separated based on the different mass-to-charge ratio, then detected by the highly-sensitive ion detectors. By analyzing different secondary ions (7 masses in parallel), information about the identity and activity of the targeted microorganisms can be obtained.
The major steps employed in single-cell analysis by NanoSIMS include environmental and cultured samples incubated with stable or radioactive isotope (with a suitable half-life) labeled substrates under in situ or microcosm conditions, then subsamples are chemically fixed, dehydrated and resin-embedded to form thin sections for NanoSIMS. When used in combination with FISH approach, samples should be hybridized with specific oligonucleotide probes prior to NanoSIMS analysis. Particularly, when an insulating sample (i.e. most of microbiological samples) is analyzed, the surface need to be coated with a thin layer of conductive materials to prevent charging effect. By providing information on the ecophysiology of microorganisms and identifying the metabolically active single cells in complex environments, NanoSIMS could be able to directly link the microbial identity with the specific activity, which is of global importance to investigate the microbes-mediated biogeochemical cycles.
The aim of this review was to describe the basic principle of NanoSIMS, and its powerful combinations with isotopic tracers, transmission electron microscope (TEM), scanning electron microscope (SEM), fluorescence in situ hybridization (FISH), catalyzed reporter deposition (CARD)-FISH, Halogen In Situ Hybridization (HISH) to analyze single-cell microbial function and identity in the field of environmental microbiology. Our review focuses on the recent application of NanoSIMS-based methodologies combined with isotope probing method to identify and visualize the key microbial populations involving in nitrogen, carbon and sulfur cycling. Moreover, some practical considerations concerning the sample preparation and phylogenetic identification when using NanoSIMS are presented. Increasing evidence strongly suggest that NanoSIMS instruments will be powerful analytical tools for environmental microbiology, provide unprecedented possibilities and huge advantages to image metabolically active single cells within complex environments. Finally, we point out the amazing potential of NanoSIMS-based techniques to solve new problems and improve our understanding in future studies of microbial ecology.