We developed a FISH protocol (EL-FISH) to label probe-targeted cell populations in a specific manner with the halogens bromine and fluorine to enable rRNA-based cell identification by NanoSIMS. In combination with stable isotope tracer experiments, we obtained functional and phylogenetic information simultaneously from individual cells in microbial communities in a single NanoSIMS analysis.
Tyramides containing a fluorophore and halogen atoms proved to be very useful during sample preparation because they enabled direct correlation of fluorescence and halogen signals. First, samples were imaged by epifluorescence or confocal laser scanning microscopy for specific probe hybridization signal. Fluorescence images obtained from microbial populations and single cells helped guide subsequent NanoSIMS analyses to spots of interest on the silicon wafer. By analyzing fluorescence first, optimization of in situ hybridization was achieved simultaneously with sample preparation for NanoSIMS and obviated the need for additional experimental efforts.
EL-FISH offers advantages in the sensitivity of detection over standard FISH techniques. In many environmental samples, cell detection by rRNA-directed in situ hybridization is hampered by the low ribosome content of target microorganisms. When directly labeled oligonucleotide probes are used, fluorescence signal intensity and element-labeling efficiency depend on the cellular ribosome content. Using EL-FISH, deposition of fluorescent dye and element label is mediated by the catalytic activity of the probe-conjugated HRP. The enzymatic signal amplification reaction allows the adjustment of probe-conferred signal intensities by variations in tyramide concentration and incubation times. Thus, cells from oligotrophic environments, which have low ribosome contents, can effectively be enriched with halogens to allow identification by NanoSIMS. We showed that elements of relatively low abundance in biological tissue, such as bromine and fluorine (10
), can be effectively used for EL-FISH cell labeling. The EL-FISH halogen labeling takes advantage of the very high sensitivity of NanoSIMS for fluorine and bromine.
The rRNA-based element labeling of individual microbial cells for cell identification by NanoSIMS does not require the modification of existing CARD-FISH protocols. The only alteration is the use of halogen-containing tyramides. Many halogen-containing fluorescent dyes are commercially available for custom tyramide conjugate synthesis and can be used for NanoSIMS analysis. In contrast, oligonucleotide probes, which are covalently modified through the addition of halogen atoms, might exhibit an altered melting behavior, depending on the number and physiochemical properties of the added element. While hybridizations with these probes might be less challenging, their altered elemental composition will require additional controls and optimization of the hybridization conditions and is associated with lower detection sensitivity.
We conducted in situ hybridizations with pure cultures of E. coli to characterize fluorine and bromine labeling of cells by EL-FISH (Table ). The relative halogen abundance obtained after EL-FISH was high compared to the natural background of the respective halogen in nonhybridized cells (Fig. ). We also compared the elemental labeling efficiency of EL-FISH with standard FISH and showed that standard FISH resulted in lower cellular halogen abundance than EL-FISH when oligonucleotide probes with three 5Fl-dU nucleotides were used (Table ).
Successful binding of the HRP-oligonucleotide conjugate to its probe sequence-defined ribosomal target site is a prerequisite for enzyme-mediated tyramide deposition. Control hybridization experiments with 13C-enriched V. cholerae cells showed that the use of a background control probe (NON338) resulted in about 40-fold lower intracellular fluorine abundance and no detected halogen background (Table ; see also Fig. S1 in the supplemental material).
We also demonstrated that combined EL-FISH/NanoSIMS provides information on metabolic activity of single cells and offers insights into the distribution of microbial activities in and among individual cells of probe-identified populations. Our method will facilitate studies of the phenotypic response of individual cells to environmental perturbations and enable the quantitative description of cellular behavior.
EL-FISH has the potential to be extended to mRNA and protein detection. This would enable the simultaneous visualization and quantification of multiple cellular characteristics when information on transcriptional and translational activity is integrated with carbon/nitrogen distribution and cellular identification. Key enzymes of metabolic pathways can reveal a cell's principle mode of energy conservation (e.g., sulfate reduction and methanogenesis) or allow identification of the assimilatory pathway that is used for the synthesis of new biomass.
Given the high spatial resolution of NanoSIMS (~50 nm), it should be possible to study intracellular distribution and localization of cellular activity (cytoplasmic, membrane bound, and periplasmic) by isotope tracer experiments or labeling of cellular features through EL-FISH or immunostaining. The detection of multiple cell features or different 16S rRNA phylotypes at the same time may require additional elemental labels. We have successfully established the use of bromine and fluorine. Li et al. report the application of iodine (19
). Selenium, boron, gold, and silver might be other suitable elements for cellular labeling because of their relatively low natural abundance in biomass (10
). Subsequent application of different probes and substrates has been demonstrated for CARD-FISH, and a combination of the technique with standard FISH is feasible (4
). Further experiments will evaluate the use of multiple elements for simultaneous identification of different microbial populations or labeling of different intracellular features, such as mRNA and proteins, based on EL-FISH/NanoSIMS.
EL-FISH/NanoSIMS combines features of high-resolution microbial imaging techniques, stable isotope probing, and microbial identification methods that rely on molecular biomarkers. This combination of techniques will facilitate the study of hitherto uncultivated microorganisms in diverse environmental communities and will reveal insights into the interrelationships of individual microbial community members (15