Confocal Raman microscopy for assessing effects of preservation methods on symbiotic deep-sea mussel gills

Confocal Raman microscopy (CRM) is a powerful tool for biological research, which can provide information regarding the composition and distribution of biomolecules in an in situ, label-free, non-destructive manner and with high spatial resolution. Sample preservation is often an unavoidable step, especially for symbiotic deep-sea samples. Moreover, protocols for the preservation of samples for CRM have not been established and specific effects of different preservation methods on biomolecules have not been studied for relevant samples. In this study, we used deep-sea mussel Gigantidas platifrons, an ideal model in the study of deep-sea symbiosis and investigated the effect of four common preservation methods on the results of CRM imaging and signals. The methods included snap-freeze (SF), SF followed by rapid fixation in methanol (SF-MeOH), 2.5% glutaraldehyde and 2% paraformaldehyde fixation (SF-GP), and 4% paraformaldehyde and alcohol fixation (PS-PA). The results of this study indicate that SF was the most effective method for the comprehensive analysis of the biomolecular composition although the sectioning success rate was relatively low. Moreover, SF-MeOH was found to be effective when SF is not sufficient in obtaining good morphology in sections, or when the effect of chemical bonding on the composition of biomolecules upon SF-MeOH can be neglected. Finally, SF-GP and PS-PA were found to be the most effective methods considering the overall morphological observation. However, they were less suitable for metabolic studies. We believe our results can provide guidance for further studies of Raman on symbiotic deep-sea biological samples. It is of great importance for the wide application of Raman technique.