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.
Scanning electron microscopy, as a surface imaging technique, is predestined for stereoscopic viewing of three dimensional objects. Generally the parallax needed for stereo impression is obtained by acquisition of two images from two different viewing angles. These images can then be examined by stereoscopic aids like mounted stereo pairs, anaglyph glasses or 3D computer monitors. The major disadvantage of this conventional approach is the prolonged acquisition and post processing time, needed for tilting and refocusing the specimen and for matching the stereo pair. After acquisition the two images must be aligned, which actually requires the exact determination of the tilt axis but in practice is commonly done "by hand" with the help of Photoshop or specific Software. Moreover, in case of sensitive specimens it is not possible to obtain the second image due to electron damage. Furthermore, usually only a subset of the images is acquired from two viewing angels during a microscope session because of the considerable effort discussed above. Live time imaging can also only be realized with the higher cost of a second detector.Depending on the type of detection and the properties of the specimen single images from scanning electron microscopy contains depth information [3]. We developed an ImageJ plugin to extract this depth information from a single picture and present it with the help of an autostereoscopic computer monitor or the anaglyph technique. For this purpose a synthetic second image is calculated and aligned to the original. The two different images can be viewed with stereoscopic aids like red-green glasses or for example with the Sharp LL-151-3D Monitor. The image quality could be greatly improved with the application of special filters before merging both images into a stereo pair. The software was implemented in Java as a plugin for the NIH public domain image processing package ImageJ [1].Actually if an exact 3D reconstruction is not possible in some cases, the plugin turned out to be very useful as a detail enhancement tool even for TEM images. Because small changes in grey scale intensity are generally not recognized by human eye, derivative images or pseudo colour rendition are often used for detail enhancement [2]. We propose our ImageJ plugin as an alternative visualization method, because faint grey scale intensity changes are placed in different z-planes and therefore easier to distinguish.
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