FIB-SEM Tomography of Biological Samples: Explore the Life in 3D
Kizilyaprak. C 1, De Bellis.D 1, Blanchard.W 1, Daraspe J 1, Mucciolo A 1 and Humbel. BM 1
1 EMF, Electron Microscopy Facility, University of Lausanne, Biophore, Quartier Sorges, 1015 Lausanne, Switzerland
Three-dimensional (3-D) spatial distribution of organelles within cells at nanometer resolution is essential to better understand cellular processes and functions. The perfect combination of focused ion beam (FIB) milling and block-face imaging in the same scanning electron microscope (SEM) constitutes one of the more encouraging methods to obtain volume information of wide variety of biological samples (Knott, Marchman et al. 2008; De Winter, Schneijdenberg et al. 2009; Heymann, Shi et al. 2009; Merchan-Perez, Rodriguez et al. 2009; Villinger, Gregorius et al. 2012; Beckwith, Beckwith et al. 2015; Blazquez-Llorca, Hummel et al. 2015; Bosch, Martinez et al. 2015; Cretoiu, Gherghiceanu et al. 2015). This method is commonly named FIB/SEM tomography. During FIB-SEM tomography, the FIB throws out Gallium ions towards the resin block’s surface and results in milling of the sample surface, discarding section as thin as 5 nm. Subsequent milling and imaging of the block-face creates an image stack with the possibility of small isotropic voxels (5 x 5 x 5nm) (Wei, Jacobs et al. 2012; Narayan, Danielson et al. 2014). FIB-SEM currently offers the highest voxel resolution, approaching resolution of TEM tomography, which makes it the reference volume EM method for high resolution imaging of site specific area.
Before discussing volume characteristics and biological results, the samples preparation which is essential for all 3D reconstruction efforts have to be address. At least 3 parameters have to be taken into consideration. 1- During SEM block-face imaging, back-scattered detectors are used to collect electrons and generate images. Contrast depends on the accumulation of heavy electron dense atoms on the structures of interest. High pressure freezing (HPF) combined with freeze-substitution (FS) and resin embedding constitutes the method of choice to find the best compromise between ultrastructural preservation and high contrast of cellular components. 2- Sample milling using FIB can induce surface damages. To ensure the dimensional integrity of the final volume cell, it is essential to assess the properties of resins used to embed biological specimen. 3- Using FIB/SEM to perform tomography requires observing some geometric rules in order to increase the resolution.
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.
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