Hi!
I'm looking forward to making a 3D reconstruction of tubular structures in zebrafish using FIB/SEM Microscopy.
I have the possibility of using a Lyra3 TESCAN FIB/SEM for this purpose, but our technicians don't know how to adjust the equipment and prepare our samples for this.
Does anyone have a detailed protocol or a paper that could guide us?
My first suggestion to this somewhat sensitive, and technical approach would be to discuss your ideas and this technical approach with Tuscan application specialists. They may know exactly the best approach and/or may be able to reference you to a specialist for this application. They want to sell more Fib's and, therefore, are usually happy to help with that in the back of their minds. This may involve fixation and embedment in a polymeric material, or perhaps addressing the animal directly. FIB ablation of an animal can involve differences in sensitivity to differing (fish)internal structures and may require appropriate adjustments to the FIB as one moves through the animal.
Dear Sebastían Otálora Tarazona , the advice provide by Var St. Jeor is the right one, contacting with the Tescan user service would be the first steep I would try. If your FIB is from another brand, sach as Raith, probably it would be good to contact with them too (probably the Tescan support will redirect you to them). The process for making a 3D reconstruction with this technique is destructive, and it basically consists in a FIB ablation of a thin slice of the sample and recording the cut image. This two steps must be repeated along the whole sample (or structure of interest). The thinner the slices, the better the resolution in the cut direction. FIB takes time and consume energy and ions, so you must balance the size of your sample and the number of slices to optimize time and costs. To guarantee the best possible image resolution, use the best possible image capture settings in your SEM, it takes more time but results always worth the time.
The following research paper also deals with some aspects for enhancing FIB-SEM reconstructions:
https://elifesciences.org/articles/25916
Probably Var St. Jeor could guide you better than me, but it might be that X-ray micro computer tomography (X ray micro CT ) could do the job too if you have access to such a device and the contrast among the different tissues is enough to provide a clear 3D reconstruction.
Hope this helps. Good luck with your research and my best wishes.
Thank you Saad and Manuel. Manuel is correct, CT is definitely a very real possibility (assuming slight to great x-ray density level differences). There would be no loss of material, like can occur with FIB. Plus, CT can be a non-destructive technique. Depending upon the instrument type, CT can have resolution levels of between a few micrometers, to a few nanometers. The latter are usually very limited regarding specimen size. Perhaps x-ray based Micro computed tomography is something worth considering?
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