PTA colour change, agarose and destaining

On 18/03/2019 21:38, Gonzalez, Brett wrote:

Hi Sarah…

My name is Brett Gonzalez and I am a postdoc at the Smithsonian National Museum of Natural History, previously a Ph.D. student with Katrine Worsaae. I am not sure if you remember, but last year I emailed you regarding some general advices towards integrating CT work into my research. Here at the museum we have a newly installed GE nanoCT and since the technicians are still technically new, I am hoping you could potentially assist once again with my questions.

I work on scale worms and since they soft bodied and fragile, I wanted to integrate alternative methods for scanning aside from just placing drying them or leaving in ethanol or other liquid. Several papers, including some where you have worked on, have used low-melting agarose to imbed the animals prior to scanning. The agarose percentages I have seen range from 0.5%-1.5% with very few other specifics. I have now tried twice, the most recent being with a 0.5% agarose embedded animal, and the entire pre-scan viewing is opaque or nearly. The animal cannot be seen. Can you think of anything in the embedding process that I am doing wrong that would prevent the X-rays from penetrating the agarose and the specimen? The agarose is prepared in 1% TAE buffer mixed with di-water. 

My only thoughts are that when putting the specimen in the agarose, the warm temperatures are causing the PTA to come out of the animal and disperse among the agarose. Could this be the case or is the agarose maybe wrong brand or age or something else? I would really like to use agarose so that specimens without chaetae don’t move during the long scans.

The only other question I have is that I had a specimen turn from ivory color (in ethanol) to blue/brown after a scan, but only in the portion being scanned. The specimen eventually turned back to the original ivory color upon upon placing in new ethanol. Have you seen this before and is this somewhat normal in liquid mounted specimens or is it an energy issue when running the scan? I have not been able to see any literature or mention of this either.

Sorry for such random questions but would really like to keep going with this technique in order to investigate muscular innervations in swimming scale worms and other annelids. Any help on the issue is greatly appreciated.

Thank you for your time.

Cheers,

Brett

--
Brett C. Gonzalez, PhD.
Postdoctoral Fellow

Smithsonian Institution
National Museum of Natural History

Hi Sarah & Brett,

Random questions are sometimes the best ones. Starting with the colour change: I often see PTA-stained regions change to blue-green-brown under X-rays, then revert after some time to the original whitish. I have assumed that this is caused by an oxidative change in the tungsten. I have not seen any effect on the scan quality. 

PTA can leach out into the agarose (where it can also turn green), but usually does not if the sample has been rinsed after staining. PTA binds strongly to proteins under acidic conditions, and this seems to be permanent if the pH stay low. PTA staining can be removed with a slightly alkaline buffer, or even with PBS, as the PTA polyacid molecule dissociates into smaller tungstate species at higher pH. 

Which brings us to the agarose: I always use agarose in water (usually 0.5%-1.0%), unless I want to keep the tissues in a buffer for some reason. I have seen PTA staining fade in agarose in PBS; iodine staining is OK. I use low-melting temperature agarose, so that it can cool to below 37°C before immersing the specimen (it gels around 33-35°). 

So I imagine the problem is the TAE, which has a pH of 8 or so as I recall, plus a chelating agent (EDTA). My guess is that your agarose effectively dissociated and dispersed the PTA more or less uniformly. Aqueous agarose might solve the problem. 

Another fun trick for embedding fragile samples is to use CyGel, a thermoreversible gel which solidifies at room temperature and melts in the fridge. This avoids the problem of removing agarose from delicate specimens: just wash in cold buffer or ethanol. However, it's really expensive. (http://www.biostatus.com/CyGel/)  

Another way to remove agarose is to drop 6M potassium iodide over the specimen while brushing off the agarose as it dissolves (this helped with a centipede holotype - lots of breakable legs... Akkari et al. 2018) 

Hope this helps. With your permission, I will also post you message and this reply to my blog (http://microtomography.blogspot.com/). 

Best,

Brian 

Akkari N, Ganske A-S, Komerički A, Metscher B. (2018). New avatars for Myriapods: Complete 3D morphology of type specimens transcends conventional species description (Myriapoda, Chilopoda). PLoS ONE 13(7): e0200158. 

https://doi.org/10.1371/journal.pone.0200158