Direct image-based correlative microscopy technique for coupling identification and structural investigation of bacterial symbionts associated with metazoans.

Coupling prokaryote identification with ultrastructural investigation of bacterial communities has proven difficult in environmental samples. Prokaryotes can be identified by using specific probes and fluorescence in situ hybridization (FISH), but resolution achieved by light microscopes does not allow ultrastructural investigation. In the case of symbioses involving bacteria associated with metazoan tissues, FISH-based studies often indicate the co-occurrence of several bacterial types within a single host species. The ultrastructure is then relevant to address host and bacterial morphology and the intra- or extracellular localization of symbionts. A simple protocol for correlative light and electron microscopy (CLEM) is presented here which allows FISH-based identification of specific 16S rRNA phylotypes and transmission electron microscopy to be performed on a same sample. Image analysis tools are provided to superimpose images obtained and generate overlays. This procedure has been applied to two symbiont-bearing metazoans, namely, aphids and deep-sea mussels. The FISH protocol was modified to take into account constraints associated with the use of electron microscopy grids, and intense and specific signals were obtained. FISH signals were successfully overlaid with bacterial morphotypes in aphids. We thus used the method to address the question of symbiont morphology and localization in a deep-sea mussel. Signals from a type I methanotroph-related phylotype were associated with morphotypes displaying the stacked internal membranes typical for this group and three-dimensional electron tomography was performed, confirming for the first time the correspondence between morphology and phylotype. CLEM is thus feasible and reliable and could emerge as a potent tool for the study of prokaryotic communities.