Preparation of Samples for Scanning Electron Microscopy: A Focus on Radish and Sunflower

1. Bio 270 – 2008 Acid Rain Experiment – Scanning Electron Micrographs Liz Casline, Hannah Inman, and Natasha Karim CLOVER RADISH SUNFLOWER How were these samples prepared? To view specimens in an electron microscope, they must be dry, because the instrument is under high vacuum, and they must be electrically conductive, because the electron beam is a current that would otherwise cause heat damage and surface charging. The fresh material was fixed (chemically stabilized) with 5% glutaraldehyde (in pH 7.2 phosphate buffer) at 4 C for 12 hours. The samples were then dehydrated by running them through an alcohol series (25, 50, 75, 95, 100, 100, 100% ethanol) for 4-12 hours per step. The samples were then dried using a critical point dryer using CO2. The samples are soaked in liquid carbon dioxide to flush the ethanol from the tissues, and then are brought to the critical point of carbon dioxide (where the liquid and gas phases exist in equilibrium – 42 C and 1300 PSI.) By slowly releasing the pressure while maintaining the critical temperature, the CO2 leaves the tissues without causing any deformation, that is, the dry tissue retains its life-like structure. For example, a grape prepared this way would still look like a grape at the end. An air-dried grape would become a raisin due to surface-tension effects. The dried specimens are then coated with a thin (20 nm) film of graphite (carbon) using a device called a carbon evaporator. The coating makes the specimens electrically conductive so that they won’t be damaged by the electron beam of the microscope. The specimens were viewed at 5 kV using a JEOL JSM-6100 scanning electron microscope. The images were captured using AIA2 Digital Imaging Systems software.