Zeiss/Xradia users meeting!

It's going on now!
Check out the tweets at
https://twitter.com/zeiss_micro

My ToScA talk

Here is a pdf of the slides from my ToScA presentation.

https://www.dropbox.com/s/nxql9qu5tvcky9c/Metscher_ToScA-2017c.pdf?dl=0

and the video collage is now on YouTube at

https://youtu.be/85wocaNifP4

Plant CT

Several people asked about published work on contrast-enhanced microCT for plant specimens. Here is an article from a group in Vienna, and a couple of more recent ones, as well as a couple of pictures I made using vascular contrast agents on wild and domestic cereal plants (details on request). 

Staedler YM, Masson D, Schonenberger J. (2013).
Plant Tissues in 3D via X-Ray Tomography: Simple Contrasting Methods Allow High Resolution Imaging. PLoS ONE 8(9): e75295.
http://www.ncbi.nlm.nih.gov/pubmed/24086499

Saoirse R. Tracy, José Fernández Gómez, Craig J. Sturrock, Zoe A. Wilson and Alison C. Ferguson. 2017. Non-destructive determination of floral staging in cereals using X-ray micro computed tomography (µCT). Plant Methods 13:9
https://doi.org/10.1186/s13007-017-0162-x

David Rousseau†, Thomas Widiez†, Sylvaine Di Tommaso, Hugo Rositi, Jerome Adrien, Eric Maire, Max Langer, Cécile Olivier, Françoise Peyrin and Peter Rogowsky. 2015.
Fast virtual histology using X-ray in-line phase tomography: application to the 3D anatomy of maize developing seeds. Plant Methods 11:55
https://doi.org/10.1186/s13007-015-0098-y

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.

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.

References for ToScA workshop notes

Handschuh S, Beisser CJ, Ruthensteiner B, Metscher BD. (2017).

Microscopic dual energy CT (microDECT): a flexible tool for multi-channel ex vivo 3D imaging of biological specimens

Journal of Microscopy (Oxford) 267(1): 3-26.

http://onlinelibrary.wiley.com/doi/10.1111/jmi.12543/full

Johnson JT, Hansen MS, Wu I, Healy LJ, Johnson CR, Jones GM, Capecchi MR, Keller C. (2006).

Virtual histology of transgenic mouse embryos for high-throughput phenotyping.

PLoS Genetics 2(4): e61.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16683035

Metscher BD. (2009).

MicroCT for developmental biology: A versatile tool for high-contrast 3D imaging at histological resolutions.

Dev Dyn 238(3): 632-640.

http://onlinelibrary.wiley.com/doi/10.1002/dvdy.21857/full

Metscher BD. (2009).

MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized tissues.

BMC Physiology 9: 11.

http://www.biomedcentral.com/1472-6793/9/11

Metscher BD. (2011).

X-ray microtomographic imaging of intact vertebrate embryos.

Cold Spring Harbor protocols 2011(12): 1462-1471.

http://cshprotocols.cshlp.org/content/2011/12/pdb.prot067033.full

http://www.ncbi.nlm.nih.gov/pubmed/22135670

Schulz-Mirbach T, Hess M, Metscher BD. (2013).

Sensory epithelia of the fish inner ear in 3D: studied with high-resolution contrast enhanced microCT.

Frontiers in Zoology 10: 63.

http://www.frontiersinzoology.com/content/10/1/63

http://www.frontiersinzoology.com/content/10/1/63/additional

Schulz-Mirbach T, Hess M, Metscher BD, Ladich F. (2013).

A unique swim bladder-inner ear connection in a teleost fish revealed by a combined high-resolution microtomographic and three-dimensional histological study.

BMC Biology 11: 75.

https://bmcbiol.biomedcentral.com/articles/10.1186/1741-7007-11-75

Sombke A, Lipke E, Michalik P, Uhl G, Harzsch S. (2015).

Potential and limitations of X-Ray micro-computed tomography in arthropod neuroanatomy: A methodological and comparative survey.

The Journal of comparative neurology 523(8): 1281-1295.

http://onlinelibrary.wiley.com/doi/10.1002/cne.23741/full

van Soldt BJ, Metscher BD, Poelmann RE, Vervust B, Vonk FJ, Muller GB, Richardson MK. (2015).

Heterochrony and Early Left-Right Asymmetry in the Development of the Cardiorespiratory System of Snakes.

PLoS ONE 10(1): e116416.

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0116416

The microCT methods exchange

Welcome to the next-generation MicroCT Methods Forum! Soon I will post some protocols and references here, and I invite you to do the same. 

Anyone can join and post (moderated by me, to prevent spam-based destruction of the blog). All comments are moderated also, but anyone (member or not) can comment on posts. 

Appropriate material includes methods, protocols, helpful tricks and advice, job announcements, references to new publications, and links to great pictures and movies (or scientific image data sets).