A combination of Focused Ion Beam and Array Tomography SEM strategies for targeted ultrastructural analysis of intermediate-size 3D volumes in small model organisms
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- EMAG - 3D & Tomographic Electron Microscopy
- Dr Irina Kolotueva (1)
1. University of Lausanne
Focused Ion Beam
- Abstract text
Ultrastructure volume data provides essential information for cell biology analysis. Numerous SEM-derived techniques, such as SBF, FIB, and AT, have emerged over the last decade. Fully or partially automated, these methods produce 3D TEM-like results by sequentially imaging single planes of the sample. The significant difference between the techniques lies in the way the portions of the sample are exposed for imaging. SBF and FIB are destructive techniques in which the image is processed inside the SEM chamber either by physical sectioning for the former or the gallium ion "surface shaving" in the latter. AT is a non-destructive procedure in which the sample is sectioned using an ultramicrotome and a diamond knife to transfer the sections on the large, non-conductive support. These sections are transferred into the SEM for image acquisition and can be imaged multiple times using different acquisition parameters. In addition to vEM, these "arrays" can be used for correlative light, electron microscopy, and immuno-gold labeling.
Using different acquisition parameters, the arrays can be imaged multiple times in different regions of interest while acquiring only the desired information, reducing the recording time, and making data analysis easier. AT image acquisition can be done in automatic, semi-automatic, or manual mode, however, in all cases, the alignment of the images will require significant effort. This is the principal disadvantage of the AT, compared to the in-SEM techniques, in which the acquired dataset is roughly aligned at the end of the acquisition cycle. All these methods have a similar xy-resolution, however, there is a significant difference with respect to z-resolution. Physical sectioning for FIB and AT restricts the thickness of the sample to ~50 nm, while FIB technology can provide z leaps as small as 5 nm. Eventually, each technique should be chosen based on the requirements of each experimental need to provide the best results within the most efficient time and cost frames.
This feature is crucial for analyzing small model organisms, such as C. elegans, Drosophila, Trichoplax, etc., that require acquiring a number of repeats from one or several genotypes. The nature of these samples is such that their manipulation for the regular TEM is problematic, being larger than unicellular samples. However, they are inconveniently small for the SBF, complicated to orient, and frequently it is impossible to provide a buffer zone for the test runs. FIB can be successfully used in cases when the localization of the ROI is not a problem while the importance of isotropic resolution is high. Array tomography, however, is indispensable when small structures have to be localized in a large volume. I provide several examples from different studies that use the tandem of FIB and AT to elucidate cellular information in C. elegans and Drosophila embryogenesis and tissue remodeling (1-3).
With or without subsequent rendering and image segmentation, these datasets already provide valuable information by transitioning 2D ultrastructure information to 3D. The combination of different vEM approaches can be used to address scientific questions and as a diagnostic tool.
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3. A targeted 3D EM and correlative microscopy method using SEM array tomography.
Burel A, Lavault MT, Chevalier C, Gnaegi H, Prigent S, Mucciolo A, Dutertre S, Humbel BM, Guillaudeux T, Kolotuev I.
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