The application of ESEM to biological samples

The Environmental Scanning Electron Microscope (ESEM) differs from a conventional SEM in that a differential pumping system maintains a pressure of gas (typically H2O) in thespecimen chamber whilst the gun remains at high vacuum. Ionizing collisions between electrons and these gas molecules create positive ions which drift down onto the sample neutralising specimen charge. It is therefore possible to image insulating samples without the need for metallic coating. The presence of water vapour in the chamber also means that a high relative humidity can be maintained and samples can be imaged in a hydrated state without the need for dehydration and fixation. These features suggest that ESEM could be well suited to imaging biological samples undergoing natural biological processes. We present a proof of principle study on the closure of stomatal pores in Tradescantia andersonia leaf tissue. An imaging protocol is developed and the advantages and limitations of this technique are discussed. Images of Vicia faba leaf tissue are also presented. Challenges include minimising beam damage and reconciling the need for an adequate physiological temperature and a low gas pressure favourable for imaging, with the thermodynamic constraints on achieving a high relative humidity.