Contrast staining for soft tissues

Probably the most versatile microCT contrast stain for soft tissue is diluted Lugol’s solution (aqueous iodine: Metscher 2009a,b; Degenhardt et al. 2010; Gignac et al. 2016). There is more than one formulation of "Lugol's;" the “IKI” solution I published in 2009 is actually 20% Lugol’s, and my “10% IKI” is 2% Lugol’s. Always be clear about the actual concentrations of iodine and iodide you are using (don't rely upon the term "Lugol's" to specify the composition unambiguously).

IKI  (Metscher 2009a, b)

2% (w/v) potassium iodide (KI) + 1% iodine (I2) aqueous solution. e.g. dissolve 2.0 g KI in 100ml distilled water, and then add 1.0 g I2 (elemental iodine, "iodine metal"). The iodide dissolves easily, and elemental iodine only dissolves in water along with iodide. Keeps indefinitely at room temperature as far as I know.

Fix fish, embryos, or whatever in your favourite aqueous fixative. Rinse samples in water or buffer (they can go directly to stain solution). Stain overnight or longer. Change the solution when it looks thinner. Wash in water.  Some iodine will still leach out; this is usually not a problem. Can be scanned in water or buffer, or mounted in agarose.

Note that some plastics (notably pieces of sponge used for bracing the specimen) will absorb some iodine, but not usually enough to be a problem for scanning.

For specimens already stored or fixed in alcohol, an alcoholic iodine solution works well. This stain is especially good for arthropods.

I2E, I2M  (Metscher 2009a, b)

1% (w/v) iodine metal (I2) dissolved in 100%  ethanol (I2E) or methanol (I2M) I2 dissolves readily in alcohol.

Take samples to 100% alcohol. Stain overnight or days or even weeks for larger specimens. Rinse in alcohol. Scan in alcohol.

Iodine does not seem to work in 70% alcohol, only 0 or 100. Anyone know why? In fact, storage in 70% ethanol usually removes most of the iodine staining.

PTA is my other favourite stain. Used in some standard histological staining methods, PTA is known to bind proteins and is commonly used in electron microscopy. The chemistry of phosphomolybdic acid appears (to me anyway) to be mostly similar, and PMA gives good staining also. And it's green. I have had some preference for PMA as a counterstain in dual-energy imaging for differentiating materials in a sample (see Handschuh et al. 2017).

PTA, PMA  (Metscher 2009a, b; Metscher 2011)

Works well on tissues fixed in formalin, 4F1G, glyoxal, Bouin’s, or alcoholic Bouin’s.

Make a 1% (w/v) phosphotungstic acid solution in distilled water.  Mix 30 ml 1% PTA solution + 70 ml absolute ethanol to make 0.3% PTA in 70% ethanol. Keeps indefinitely. The pH must be on the acidic side: the above solution comes out around 2.9. The affinity of PTA for different proteins is pH-dependent (Nemetschek 1979, Scott 1971, Silverman 1969), but I have not tested this systematically for use in microCT imaging.

Take samples to 70% ethanol. Stain overnight or longer. Change periodically for larger samples. Change to 70% ethanol. Scan samples in 70% - 100% ethanol. Store in 70-100% ethanol.

Gives high general radiopacity and excellent contrast among tissues and structures in vertebrate embryos and soft-bodied invertebrates. Penetration is vaguely 1-2 mm per day, so overnight is usually sufficient for samples no thicker than about 3-4mm.   Staining is stable for months if not years. Samples can be embedded and sectioned for histology afterward.

Osmium tetroxide (Johnson et al. 2006; Metscher 2009a, b)

Standard EM post-fixation, binds abundantly to lipids.

Same as routine EM processing. Osmium-stained samples can be scanned in resin blocks, with some loss of contrast.

Gives very high radiopacity and no better tissue contrast than PTA. Osmium tetroxide is volatile and toxic, but your local EM lab is probably already set up to work with it.