Abstract
BackgroundIt 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.ResultsAll 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.ConclusionsThis 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.
Highlights
It is well known that preparation of biological 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
To identify a robust SEM preparation method that caused the least modifications to tissue dimensions and morphology we compared the effects of seven different fixation protocols on the preservation of leaf samples from Arabidopsis thaliana, barley and cotton
Methanol fixation was followed by either further dehydration in methanol and critical point drying in methanol (‘Methanol’), or by ethanol dehydration and critical point drying in ethanol (‘Methanol-ethanol’)
Summary
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. A well-known artefact of preparing biological tissue for Scanning Electron Microscopy (SEM) is the shrinkage of tissue during fixation, dehydration and critical point drying (CPD) steps In the past, these artefacts were documented largely by comparing the surface morphology of animal and some plant tissues prepared using different protocols. Boyde and Boyde (1980) showed that unfixed potato tuber tissue processed through dehydration and CPD shrank less (15%) than glutaraldehyde fixed tissue (30%; [2]) Another source of shrinkage was the critical point drying process and postCPD storage, which could be due to retention of water or ethanol in the tissue after drying, which slowly evaporated during storage [2,5]
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