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.
