Fluorescent in situ hybridization (FISH) has been a powerful technique for assessing microbial communities in the environment.  In a FISH experiment, fluorescently labeled oligonucleotide probes are designed to hybridize to target-specific regions of ribosomal RNA (rRNA) molecules, and successful hybridization reactions indicate the presence of microbial groups of interest.  In contrast to DNA sequencing-based assays, FISH indicates whether cells are alive and/or active because active cells tend to produce more FISH probe targets, rRNA molecules.  In addition, FISH provides information about cell abundance without influence of rDNA copy number in target organisms and PCR amplification biases.  Analysis of FISH images, however, can be complicated by inability to distinguish probe-conferred fluorescence and background/autofluorescence.  In this study, we utilized spectral imaging technology to identify and confirm the presence of probe-conferred fluorescence in FISH images.  Because spectral imaging enables detection of several distinct spectral signatures that are present in each cell, each target cell was simultaneously labeled with a general bacterial probe and two group-specific probes.  In following experiments, we simultaneously applied probes against multiple target groups to achieve differentiation of a larger number of distinct target groups in a single FISH experiment.  In addition, we developed an image processing protocol that provides quantitative information for target bacterial groups.  Our protocol was applied to environmental samples, including seawater collected in a local salt marsh.                

Results/Conclusions

Analysis of fluorescent spectra within each cell enabled differentiation of probe-conferred spectra from background and autofluorescence.  Detection of multiple probe-conferred spectra in each target cell also increased confidence in our results.  Spectral imaging combined with our image analysis protocol enabled quantification of bacterial groups that are as rare as 1% in the total community in seawater.  Furthermore, simultaneous application of probes against multiple different bacterial groups and subsequent separation of fluorescent spectra enabled differentiation of more than ten different target groups in a FISH experiment.