Three-dimensional (3-D) spatial distribution of organelles within cells at nanometer resolution is essential to better understand cellular processes and functions. Currently, FIB-SEM tomography provides a promising technology to generate volume data with nanometer resolution (Bushby et al., 2011; Heymann et al., 2009; Knott et al., 2011; Villinger et al., 2012; Wei et al., 2012). A FIB-SEM microscope is a scanning electron microscope (SEM) combined with a focused ion beam (FIB) where both beams coincide at their focal points. This combination enables to locally section bulk embedded-resin samples by ion milling, creating a fresh surface for subsequent imaging with the electron beam. This process is repeated automatically to generate 3-D information of relatively large volumes with a field of view of several micrometers.

Any stained and embedded resin samples prepared for transmission microscope can be used for FIB-SEM tomography offering the possibility to visualise directly in 3D a wide range of biological samples and typically volumes with a pixel size of 10 nm can be achieve (Figure1). However, to obtain a better resolution, considerations have to be given concerning ultrastructure preservation and samples preparation (Kizilyaprak et al., 2015). High pressure freezing (HPF) combined with freeze-substitution (FS) and resin embedding constitutes a method of choice to find the best compromise between ultrastructural preservation and high contrast of cellular components (Figure2).

In conclusion, we propose biological sample preparation protocols that can serve as starting point to visualize in 3-D wide range of biological samples at nanometer resolution including HPF/FS samples and correlative microscopy approach using FIB-SEM Tomography.

References:

Bushby, A.J., P’Ng K, M., Young, R.D., Pinali, C., Knupp, C., Quantock, A.J., 2011. Nat Protoc 6, 845-858.

Heymann, J.A., Shi, D., Kim, S., Bliss, D., Milne, J.L., Subramaniam, S., 2009. Journal of structural biology 166, 1-7.

Kizilyaprak, C., Longo, G., Daraspe, J., Humbel, B.M., 2015. Journal of structural biology 189(2), 135-146.

Knott, G., Rosset, S., Cantoni, M., 2011. J Vis Exp, e2588.

Villinger, C., Gregorius, H., Kranz, C., Hohn, K., Munzberg, C., von Wichert, G., Mizaikoff, B., Wanner, G., Walther, P., 2012. Histochem Cell Biol 138, 549-556.

Wei, D., Jacobs, S., Modla, S., Zhang, S., Young, C.L., Cirino, R., Caplan, J., Czymmek, K., 2012. Biotechniques 53, 41-48.

Figures:

Figure 1 : Electron Micrograph of chemically fixed biological samples in FIB-SEM at 2kV accelerated voltage using back-scattered electron detector. A) Macrophage cells co-infected with HIV and Salmonella (Horizontal Field of View (HFW) = 30µm, Pixel Size (PS) = 7.3nm). B) Liver cells from mouse (HFW = 12 µm, PS = 2.9 nm). C) Roots of Arabidopsis Thaliana (HFW = 55µm, PS = 13.4nm).

Figure 2 : Electron Micrograph of HPF/FS liver samples in FIB-SEM at 2kV accelerated voltage using back-scattered electron detector (HFW = 8.5µm, PS = 2.9nm).

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