Cryogenic FIB Lift-out as a Preparation Method for Damage-Free Soft Matter TEM Imaging

The removal of a thinned lamella from a bulk sample for Transmission Electron Microscopy (TEM) analysis has been possible in the Focused Ion Beam Scanning Electron Microscope (FIB-SEM) for over 20 years either via in situ (by use of a micromanipulator) or ex situ lift-out approaches [1]. Both offer swift, site specific preparation for TEM analysis, particularly in light of advancements in corrected TEM. These techniques, however, are currently only applied to samples at room temperature, typically from the materials sector. The majority of biological samples contain a high degree of water, which will be removed under vacuum, leading to the shrinking and rearrangement of the sample. To overcome this, samples can be prepared by either critical point drying, fixation and resin impregnation (often combined with heavy metal staining) or cryogenic fixation. For both fixed and cryo-preserved samples, the preparation of thin-sections has always typically been prepared with a microtome, which yield samples of 60100 nm. However, these commonly suffer from compression artifacts and/ or knife marks, which distort data. There is also a desire to move away from staining and methods which dehydrate or allow / permit structural or chemical re-arrangement.