Cryo-laser scanning confocal microscopy of diffusible plant compounds

BACKGROUND: The in vivo observation of diffusible components, such as ions and small phenolic compounds, remains a challenge in turgid plant organs. The analytical techniques used to localize such components in water-rich tissue with a large field of view are lacking. It remains an issue to limit compound diffusion during sample preparation and observation processes. RESULTS: An experimental setup involving the infusion staining of plant tissue and the cryo-fixation and cryo-sectioning of tissue samples followed by fluorescence cryo-observation by laser scanning confocal microscopy (LSCM) was developed. This setup was successfully applied to investigate the structure of the apple fruit cortex and table grape berry and was shown to be relevant for localizing calcium, potassium and flavonoid compounds. CONCLUSION: The cryo-approach was well adapted and opens new opportunities for imaging other diffusible components in hydrated tissues.