University of Rochester
In this project, samples I choose to observe are Arborvitae "Woodward Globe" and a juniper which are common in Rochester, and their leaf images are shown in Figure 1. Two pieces of leaves are cut from both two evergreen needles respectively, to prepare samples for SEM imaging. All samples are about 1-2cm long.
Scanning electron microscope(SEM) operates at high vacuum to avoid gas molecules interfering with primary electron beam and secondary or backscattered electrons emitted from the sample. Water in biological materials make them incompatible with high vacuum system since water vaporization will distort electron beam and degrade images. Dehydration is a crucial step in biological sample preparation.
There are three common drying methods, including critical point drying(CPD), HMDS(hexamethyldisilazane) drying and air drying. For air drying process, collapse and distortion of surface structures will take place due to high surface tension between the interface of air and water. CPD and HMDS drying are both effective techniques to obtain dried biological sample with intact surface structure. In addition, compared with expensive and time-consuming CPD technique, HMDS drying is much easier, cheaper and faster.
The specimen was immersed in varying concentrations of ethanol, culminating in complete replacement of water in the sample. Then ethanol is removed HMDS through a graded series of HMDS-ethanol mixtures. After drying overnight in fume hood, completed dried samples are attained.
Normally, when electron beam strikes a conductor, the electrical potential will be dissipated, and charges will be conducted through the specimen and be grounded by contacting with the specimen stage. However, as evergreen needles are insulators, which indicates that they cannot dissipate excess negative charges and then localized charges build up. Charging artifacts will appear and cause deflection of electrons, which result in distortion of sample image and degrading the image quality. Specimen coating serves an important role in eliminating charging effect. It enables to increase conductivity of specimens, reduce thermal damage, increase secondary and backscattered electron emission, and increase mechanical stability. Sputter coater is an effective technique to cover the sample with a thin conformal conductive layer. In this project, all sample are coated with a 6nm-thickness gold layer.