Most samples are not suitable to be analyzed with a Scanning-Electron-Microscope (SEM) in their natural state. Instead, they require a series of time-consuming preparational steps prior to SEM analysis. This is especially true for medical and biological samples, which are usually non-conductive material and often contain a very high percentage of water. For example the water content of a fresh salad leaf makes up 98% of its total weight.
To prevent the accumulation of static electric charges during the exposition to the high-energy electron beam, non-conductive materials are normally sputtered with a very thin layer of conductive material (gold, gold/palladium, platinum, etc) to maximize signal output and improve spatial resolution. The instrument is called a Sputter Coater.
Wet sample preparation
The ideal sample is completely dry during sputtering and SEM analysis. Hard and dry materials such as wood, bone, feathers or shells need only little further treatment compared to living cells, delicate animal tissues, or soft-bodied organisms.
Fast and simple air-drying is no option for the preparation of e.g. the fresh salad leaf, because it would cause shrinkage and collapse of living cells. Instead, most biological and medical samples require a special chemical treatment for stable and state-of-the-art preservation. After an initial series of chemical fixations, each specimen has to be dehydrated step-by-step by solvents such as ethanol or acetone . After complete dehydration, the formerly wet sample undergoes critical-point-drying (CPD). This method replaces dehydration solvents (ethanol or acetone) with a transitional fluid, such as liquid carbon dioxide. The procedure takes place at high pressure in a special instrument (called a ‘Critical Point Dryer‘) and ensures only minimal or no change of the original (living) state.
Continue reading how the original SEM scans are captured: Scanning-Electron-Microscopy (p. 3/4)