Table 2.

Summary of sample preparation techniques

Preparation	Protocol	Considerations
Drying	Air drying, HDMS or critical point drying (for cells) and freeze drying (for tissues) [53]	• Samples become very delicate • Subsequent fixation not possible • Long or thin parts prone to movement during scanning • Compatible with electron microscopy in correlative imaging
Chemical fixation	10% neutral buffered formalin [54] 1% glutaraldehyde in acetone [55] Copenhagen mix (for plants: 70% absolute alcohol, 2% glycerol, 28% water) [41]	• Typically used before heavy metal staining to minimise sample shrinkage • Imaging may be performed in liquid or air (typically ethanol or distilled water) • Sample may move during scanning; measures to prevent movement such as packing with foam are advised • If imaging in air, measures to prevent drying are advised (e.g. placing sample in sealed container with a small reservoir of liquid to maintain humidity)
Embedding	Resin [56] or wax [34]	• Effective at preventing sample movement • Good for samples with long or thin parts which may otherwise vibrate during scanning, causing blurred imaging • Can be used with or without staining • Resin compatible with block face serial sectioning for correlative imaging
Freezing	Freezing [57] or vitrification [19]	• Use of a cryo-stage is necessary during CT imaging of frozen samples • Vitrification is typical for soft nCT as it minimises cryo-damage
Native tissue	No fixative, with staining [26]	• Used to provide contrast whilst minimising change in tissue mechanical properties (iodine in phosphate buffered saline has been used for this purpose)
	No fixative, no staining [58]	• Fix and stain unsuited to some tissues due to uptake of fluid causing swelling, e.g. intervertebral disc