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 | • 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 | • 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 |