pressure of gas, and of the aperture diameter used. This distance varies from around 10 mm to a fraction of a millimeter as the gas pressure may vary from low vacuum to one atmosphere. For optimum operation, both the manufacturer and the user must conform, in the design and operation, to satisfy this fundamental requirement. Furthermore, as the pressure can be brought to a very low level, an ESEM will revert to typical SEM operation without the above disadvantages. Therefore, one may trade-off the ESEM characteristics with those of SEM by operating in a vacuum. A reconciliation of all these disadvantages and advantages can be attained by a properly designed and operated universal ESEM. Concomitant with the limitation of useful specimen distance is the minimum magnification possible, since at very high pressure the distance becomes so small that the field of view is limited by the PLA1 size. In the very low magnification range of SEM, overlapping the upper magnification of a light microscope, the superior field is limited to a varying degree by the ESEM mode. The degree of this limitation strongly depends on instrument design. As X-rays are also generated by the surrounding gas and also come from a larger specimen area than in SEM, special algorithms are required to deduct the effects of gas on the information extracted during analysis. The presence of gas may yield unwanted effects in certain applications, but the extent of these will only become clear as further research and development is undertaken to minimize and control radiation effects. No commercial instrument is as yet (by 2009) available in conformity with all the principles of an optimal design, so that any further limitations listed are characteristic of the existing instruments and not of the ESEM technique, in general. Transmission ESEM The ESEM can also be used in transmission mode (TESEM) by appropriate detection means of the transmitted bright and dark field signals through a thin specimen section. This is done by employing solid state detectors below the specimen, or the use of the gaseous detection device (GDD). The generally low accelerating voltages used in ESEM enhance the contrast of unstained specimens while they allow nanometer resolution imaging as obtained in transmission mode especially with field emission type of electron guns. ESEM-DIA ESEM-DIA is an abbreviation standing for a system consisting of an ESEM microscope coupled to a digital image analysis (DIA) program. It