ESA-SRB-AOTA 2019

FIB-SEM tomography is a new developing method, which has opened a new era in electron microscopy – it makes possible to visualize internal three-dimensional organization of biological structures with nanometer scale resolution [1]. This method is very promising for spermatogenesis studies. Indeed, intercellular interactions and complex microtubules machinery can be revealed with high three-dimensional resolution [2].  

Proper sample preparation has a crucial importance for preservation of embedded tissue ultrastructures. In this study high-pressure freezing was used as the most delicate method of tissue preservation. The protocols of freezing and freeze-substitution have been optimized for seminiferous tubules preparation. It was shown that tannic acid-mediated osmium impregnation [3] dramatically improves contrast of tissue structure. After freeze-substitution tissue were embedded in Epon resin and examined with FEI Helios DualBeam plasma FIB with Auto Slice & View software, voxel size up to 5 x 5 x 5 nm.   

2D images were segmented with iLastic software, 3D structures were rendered with FEI Amira platform. Sertoli cells spatial organisation, germ cell development and three-dimensional microtubules distribution in the cells have been shown in mouse. 

References:

1. Kizilyaprak, Y.-D. Stierhof, B.M. Humbel, Volume microscopy in biology: FIB-SEM tomography // Tissue and Cell, 2019, V. 57, pp. 123-128.
2. Luckner, G. Wanner, Precise and economic FIB/SEM for CLEM: with 2 nm voxels through mitosis // Histochem. Cell Biol., 2018, V. 150(2), pp. 149-170.
3. Jiménez, K. Vocking, E.G. van Donselaar, B.M. Humbel, J.A. Post, A.J. Verkleij, Tannic acid-mediated osmium impregnation after freeze-substitution: A strategy to enhance membrane contrast for electron tomography // J. of Struct. Biol., 2009, V. 166, pp. 103-106.