Methanol fixation of plant tissue for Scanning Electron Microscopy improves preservation of tissue morphology and dimensions

BACKGROUND: It is well known that preparation of biological (plant and animal) tissues for Scanning Electron Microscopy (SEM) by chemical fixation and critical point drying results in shrinkage of tissues, often by up to 20-30%, depending on the tissue type and fixation protocol used. We sought to identify a protocol that would preserve tissue size and morphology better than standard chemical fixatives and dehydration regimes. We compared a range of processing techniques by quantifying changes in tissue size and recording details of surface morphology using leaf tissues from three commonly studied species; Arabidopsis thaliana, barley and cotton. RESULTS: All processing protocols altered tissue dimensions. Methanol fixation and dehydration, followed by a further short (1 h) dehydration step in ethanol and critical point drying (which was based on a previously published method), preserved tissue dimensions most consistently of all protocols tested, although it did cause 8% shrinkage in all three species. This protocol was also best for preservation of surface morphology in all three species. We outline a recommended protocol and advise that the method is best trialled for different tissues, especially thicker or larger samples. CONCLUSIONS: This study shows that simultaneous fixation and dehydration in methanol followed by ethanol results in better preservation of dimensions and morphology of critical point dried plant tissues than other fixation and dehydration procedures. It is a quick and simple method, and requires standard SEM preparation equipment.