ToScA Workshop (Life Sciences): Microtomography for life sciences research

This workshop is offered twice on Wed. 6 Sept. and will introduce some methods for enhancing x-ray contrast in non-mineralised tissues and techniques for mounting biological samples for microCT imaging, especially embryos and other soft tissues, insects and other invertebrate specimens, and any samples of particular interest to the participants.

We will begin with some principles of x-ray imaging, discuss various types of samples and applications, and then work with your own interesting specimens.

You are invited and encouraged to bring your own samples to the workshop! Fixation and staining can take days, so you might want to prepare your samples ahead of time.

Please also bring pertinent questions, including issues concerning image analysis, publishing, and archiving!

Details of stains etc. are given in the accompanying blog entry. And you may of course email me with questions: brian.metscher@unvie.ac.at

1) General advice about sample preparation for microCT imaging  

Fixation:

The best fixation for microCT is the one that preserves the features you need to see. I have had good results with most of the common fixation procedures, but the properties and effects of the fixative must be taken into account for contrast staining. Usually most relevant are shrinkage, decalcification, removal of lipids or carbohydrates, and protein condensation or precipitation.

Preservation:

Samples can usually be stained effectively after storage in 70% ethanol or in formalin. If you want to use an aqueous stain, transfer the sample back to an aqueous solution; likewise for alcoholic stains. Dry samples are easy (they're dry).

2) Some tips for preparing different sample types

Insects & other arthropods:

I have had good results from alcohol-fixed crawlies by re-fixing them in alcoholic Bouin's (1:1 Bouin's:ethanol/IMS) for a few hours or longer and then dehydrating to ethanol (absolute but not anhydrous, i.e. 96-100%), followed by staining in I2E (below).

Others have made excellent images of critical-point dried insects (better than HMDS; Sombke et al. 2015).

Dry insects can be scanned easily, but the internal anatomy is dodgy. Chitinous structures usually come out beautiful. Pins can be a challenge, but scans can be done with pinned insects.    

Embryos and other squishy samples:  

My favourite fixative for soft stuff is 4F1G (4% formaldehyde and 1% glutaraldehyde in phosphate buffer, or other appropriate buffer, like PBS, or whatever your samples are happy in). The actual concentrations of the two fixatives are not crucial; I usually just add glutaraldehyde to 10% NBF and I'm done. The glut must be high-grade and fresh - otherwise it polymerises and becomes less effective.

PTA and iodine both give good results. Shrinkage can be a problem...   

Little fish and anything that sort of resembles a little fish:  

Mostly the same as embryos, but pay attention to whether you want to see e.g. brain, visceral organs, muscles, bones, teeth, etc. Staining with PMA can allow nice distinction of mineralised tissues and soft tissues; PTA in methanol has given good images of hearing structures (Schulz-Mirbach et al. 2013a, b).

Samples in methanol:

Samples preserved for nucleic acids work are often stored in methanol, typically after aldehyde fixation. These can be stained easily and effectively with  PTA in absolution methanol (van Soldt et al. 2015).

3) Mounting samples for microCT

The sample must be immobilised and held on a vertical rotation axis for the duration of the scan. I often use 0.5-1.0% agarose to embed (not infiltrate) samples in narrow plastic tubes or micropipette tips. Other schemes can work also, and may work better for some kinds of objects: bits of sponge and soda straws have helped on occasion.  Other friends of sample mounting include Legos, Parafilm, UHU Patafix (Blu Tack), and a hot-glue gun.