Transmission-mode imaging in the environmental scanning electron microscope (ESEM)
Electron microscopy was first conducted in the 1930s with the advent of the TEM and later the STEM. In 1969, the first commercial SEM was released, with the possibility of retrofitting it to behave like a STEM following soon afterwards. In 1979, Danilatos and Robinson advanced electron microscopy by creating a new type of SEM which allowed a controlled quantity of gas into the sample chamber, termed ESEM. The most recent evolution in this line was the combination of ESEM and STEM in 2005, a procedure termed Wet STEM. The focus of this work is on investigating applications of this new technique, along with the contrast mechanisms involved in forming an image. To that end, a wide variety of samples will be imaged. Clay and paint suspensions (colloids) are used to test Wet STEM’s capacity to image submerged objects, as well as thin objects which are stacked together. Diblock copolymer films are used to test Wet STEM’s ability to distinguish chemically similar materials without staining, the physical effects of heavy metal staining and to demonstrate the necessity of gas for the purpose of charge neutralisation. Single cell biological samples are also investigated. Internal contrast in mammalian cells is visible without recourse to staining, but chemical fixation is required despite maintaining a high relative humidity. Bacteria are more resilient and as such are easier to image than animal cells, requiring no prior treatment. When exposed to low relative humidity, bacteria are found to collapse. The collapse pattern is observed to differ between wild-type and cytoskeletal-deficient bacteria of the same species and strain, so it is likely that dehydration-induced collapse offers information about the position and shape of the bacterial cytoskeleton.