I want to embed a 2 mm thick tissue sample (with a polymerized diaminobenzidine stain) into glycol methacrylate and use synchrotron phase contrast x-ray microtomography to image the tissue. However, I am not sure whether glycol methacrylate is translucent to x-rays at wavelengths ranging from 15 to 30 keV. Could someone please let me know if they have ever had success with imaging through this material via x-rays?
In addition, I was wondering if the x-ray phase decrement for polymerized diaminobenzidine is distinct enough from that of glycol methacrylate plastic that it should give good contrast. Thanks!
Dear Logan Thrasher Collins ,
knowing the chemical formula and the density one can easily calculate the x-ray transmission properties of a sample by using the NIST/XCOM database:
https://physics.nist.gov/PhysRefData/Xcom/html/xcom1.html
Taking the formula C10H14O4 for glycol methacrylate and a density of 1g/cm³ I have listed the linear x-ray attenuation coefficients µ for some x-ray photon energies and some sample thicknesses d in the attachment.
If glycol methacrylate plastic has significant different formula and density you may calculate the µ by yourself.
For small sample thickness in the low cm range the glycol methacrylate is quite translucent...
With respect to the x-ray phase decrement delta, you know, that delta is proportional to the density. I do not know the density of your stain (polymerized diaminobenzidine). So you may estimate by yourself whether one should be able to differentiate your stain from the glycol methacrylate plastic.
Best regards
G.M.
Dear Logan Thrasher Collins ,
let me come up with an additional remark;
you have 'stained' your tissue sample, and that (2mm) sample will have density fluctuations by itself.
So, how do you think to discriminate the stain distribution from the inherent density distribution of the tissue matrix?
Adding some (optical) stain onto/into the sample having about the same density (stain compared to tissue sample) will, to my opinion, not very effective in the x-ray domain. In the x-ray regime you see the density distribution when dealing with transmission and density gradients when dealing with x-ray phase contrast techniques; attenuation and phase decrement are proportional to density.
The glycol methacrylate around the tissue sample will be homogenous; it will not affect the sample image with respect to the image contrast; unless it penetrates into the tissue sample...
Best regards
G.M.
Gerhard Martens thank you for your helpful replies - I should note that I am working with expanded tissue (ExM), so the density of the tissue itself will be very low. Since low density tissue will not show up to x-rays, I am planning to use a method called Unclearing (see Preprint Unclearing Microscopy
and https://www.panluminate.com/processing-services) to 'rebuild' the morphology of the tissue. Unclearing anchors horseradish peroxidase (HRP) onto primary amines in the tissue. This allows redox-based deposition of diaminobenzidine precipitates or other reagents in a localized fashion.My main point of concern is whether the diaminobenzidine precipitates formed in place of the expanded cells will show up against a glycol methacrylate background. If you have any further comments/insights, please do let me know!
Very much appreciated!
Logan
Thanks for your email I am not the person who wrote this article I think this is another person whose name just same like me
I am not sure it is releveant, just to note : one can ultrasonic (for ~1mm size water density objects) or electrodynamic (for 0.01..0.1mm size ) levitation to put sample into synchtron X-ray beam. E.g.
https://www.diamond.ac.uk/Science/Research/Tech-Updates/2019/22-10-2019.html
https://iopscience.iop.org/article/10.1088/1742-6596/2380/1/012112/pdf
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