Scanning Electron Microscope - Sample Preparation

Sample Preparation

All samples must also be of an appropriate size to fit in the specimen chamber and are generally mounted rigidly on a specimen holder called a specimen stub. Several models of SEM can examine any part of a 6-inch (15 cm) semiconductor wafer, and some can tilt an object of that size to 45°.

For conventional imaging in the SEM, specimens must be electrically conductive, at least at the surface, and electrically grounded to prevent the accumulation of electrostatic charge at the surface. Metal objects require little special preparation for SEM except for cleaning and mounting on a specimen stub. Nonconductive specimens tend to charge when scanned by the electron beam, and especially in secondary electron imaging mode, this causes scanning faults and other image artifacts. They are therefore usually coated with an ultrathin coating of electrically conducting material, deposited on the sample either by low-vacuum sputter coating or by high-vacuum evaporation. Conductive materials in current use for specimen coating include gold, gold/palladium alloy, platinum, osmium, iridium, tungsten, chromium, and graphite. Additionally, coating may increase signal/noise ratio for samples of low atomic number (Z). The improvement arises because secondary electron emission for high-Z materials is enhanced.

An alternative to coating for some biological samples is to increase the bulk conductivity of the material by impregnation with osmium using variants of the OTO staining method (O-osmium, T-thiocarbohydrazide, O-osmium). Nonconducting specimens may be imaged uncoated using Environmental SEM (ESEM) or low voltage mode of SEM operation. Environmental SEM instruments place the specimen in a relatively high-pressure chamber where the working distance is short and the electron optical column is differentially pumped to keep vacuum adequately low at the electron gun. The high-pressure region around the sample in the ESEM neutralizes charge and provides an amplification of the secondary electron signal. Low-voltage SEM is typically conducted in an FEG-SEM because the FEG is capable of producing high primary electron brightness and small spot size even at low accelerating potentials. Operating conditions to prevent charging of non-conductive specimens must be adjusted such that the incoming beam current was equal to sum of outcoming secondary and backscattered electrons currents. It usually occurs at accelerating voltages of 0.3-4 kV.

Embedding in a resin with further polishing to a mirror-like finish can be used for both biological and materials specimens when imaging in backscattered electrons or when doing quantitative X-ray microanalysis.

The main preparation techniques are not required in the environmental SEM outlined below, but some biological specimens can benefit from fixation.