FIB-SEM tomography is a new powerful tool for visualization of various biological structures which provides three-dimensional information with nanometer scale resolution . Sample preparation for FIB-SEM is almost the same with the cells and tissue preparation for conventional TEM, except the slicing.
Spermatogenesis is the process by which spermatogenic stem cells undergo division and differentiation to produce spermatozoa. Microtubules having a crucial role in this process . FIB-SEM tomography provides unparalleled information about the spatial distribution of microtubules in the cell, and thus, a greater understanding of intracellular processes. The possibility of reconstruction of microtubules spatial distribution has been shown for different cells involved in spermatogenesis: Sertoli cells, all phases of germ cell development and mature spermatozoa.
In comparison with conventional SEM, FIB-SEM tomography provides not only exterior morphology of the cell but also detailed visualization of internal structures. Detailed 3D reconstruction (resolution up to 5 x 5 nm/pixel and section thickness of 20 nm) was performed for mouse sperm. FEI Helios DualBeam plasma FIB with Auto Slice & View software was used (operating 2 kV for backscattering electron imaging and 30 kV for oxygen FIB milling).
Ion beam interaction with embedding medium is of crucial importance in the FIB-SEM technique. Three embedding mediums were studied: Epon, LR White and Lowicryl HM20. The best results have been achieved with Epon resin.