Monitoring and Analysis of the Environment for Biomaterials in the ESEM Under “Wet Mode” Conditions

Abstract

The use of pressure limiting apertures and differential pumping systems has allowed the realization of scanning electron microscopes that can tolerate specimen chamber pressures in the range 1-20 torr (0.13-2.7 kPa) while maintaining the electron gun and column at high vacuum (< 10-6 mbar, 0.13 mPa) [1]. The presence of a (water)-gas atmosphere in the specimen chamber allows for imaging of uncoated biomaterial at high beam energies. In principle the wet-mode technology of the ESEM enables the investigation of fully hydrated native biological specimen. Chemical fixation, total dehydration and the respective artefacts appearing after these preparation steps for conventional SEM investigations can thus be prevented. Moreover, the period between specimen sampling and the final investigation in the ESEM can be reduced to a minimum, and theoretically the specimen can be reused or recultivated. ESEM investigations under "wet-mode" conditions are carried out in an "artificial" gas atmosphere with a partial water pressure of approximately 10 torr that is also necessary for image generation. When reaching the conditions of equilibrated humidity inside of the ESEM specimen chamber, the natural atmosphere composed of O 2 , N 2 , CO 2 and variable concentrations of water vapour is replaced stepwise by a pure (water)-gas atmosphere by means of auto-flood cycles. The measurement and monitoring system introduced here should help to analyse and understand the thermodynamic parameters t determining the environmental conditions for the unfixed and hydrated biological specimen inside the FEI XL30 ESEM vacuum chamber. Therefore we intended to monitor all sequences in the course of ESEM processing and investigation steps for bio-material under wet-mode conditions: this refers to the whole process of evacuation by dynamic automatic flood cycles, the investigation process in a relative stable humidity equilibrium and the final flood process to return to normal atmospheric conditions. The main goal of our monitoring system is to rule out process steps with the potential for producing fine structural artefacts at the hydrated specimen surface. Here, coarse and highly dynamic alterations in temperature, pressure and humidity, causing surface drying processes, have to be avoided.