In both techniques 3 µL of specimen is typically deposited on a copper grid with a hydrophilized carbon film, which serves as a support for the sample. The excess of sample is then blotted, leaving a thin film of specimen. In cryoTEM, the grid is immediately plunge-frozen in liquid ethane, at temperatures around -180 °C. The specimen is then kept in liquid nitrogen until it is inserted in the microscope and is imaged at cryogenic temperatures. In cryoTEM, the specimen is preserved in a hydrated environment, near to its native state.
Whilst in nsTEM the excess of sample is also blotted, leaving a thin film of specimen. A solution of heavy metal salt is then deposited on the support and the excess is blotted off. The grid is then inserted and observed in the microscope at room temperature. In this method, the stain acts both as a contrast enhancing agent.
The following table shows the potential of both techniques in terms of analysis:
|Type of analysis||cryoTEM||nsTEM|
|Integrity and debris||No||Yes|
|Level of aggregation||No||Yes|