Electron Tomography of Whole Mounts
tert-Butanol Freeze-Dried Colloids
Fig. 1: TEM micrographs and ET reconstruction of cobalt nanoparticle superstructures. a) The TEM micrograph of self-assembled nanoparticle superstructures. b) 3D reconstruction shows that the capsids with a hollow interior (left) and filled with organic materials (right). c) A cross-sectional view of the particles reveals that both the particles contain ~20 nm thick layered shell. See also video S1. Scale bars (a-c) corresponds to 20 nm.
Fig. 2: Comparison of AD and tBFD methods. a-c) TEM images show that under AD method colloidal particles undergo deformation and beam damage. d-f) The tBFD method prevents deformation, and the sample appears to be stable under electron beam during collection of tilt series. g-i) shows 3D reconstruction and its cross-sections. g and h reproduced with permission from ref.12. Scale bars (a-i) corresponds to 100nm. g and h reproduced with permission from ref.12,
Fig. 3: Comparison of cryo-TEM and tBFD. a) Cryo-TEM images for a self-assembled resilin-fusion protein superstructure showing flattened particle under cryo-TEM conditions. d-f) tBFD method retains the spherical nature with improved contrast. g-i) 3D reconstruction and cross-sectional view reveals that the particles are composed of ordered structures. d-f reproduced with permission from ref.13. Scale bars (a-h) corresponds to 200nm. d-f reproduced with permission from ref.13, Copyright 2018,
Rapid progress in the instrumentation, sample preparation methods, and computational power have triggered a revolution in electron tomography methods. Herein, we adapted a straightforward freeze-drying method using tert-butanol for electron tomography of whole mount colloids. This approach will overcome some of the common artifacts in electron microscopy specimen preparation.
It has been shown that in cryo-vitrification, the ice thickness varies from 70-120 nm depending on the type of TEM grid and the support film. Therefore, in cryo-TEM particles over ~200 nm thick often undergo flattening during vitrification. Therefore, to observe the unaltered structural features, the particles should be within the above-mentioned limits [9]. Thus, there is a need for other specimen preparation methods to study whole mount colloids. We have used simple tert-butanol freeze-drying (tBFD) method for TEM imaging and ET reconstruction of whole mount colloids (d > 200 nm). Further, we have compared the results obtained from tBFD method with AD, CPD, and cryo-vitrification. Our results suggest that morphological features are retained in tBFD and applicable to self-assembled synthetic and bio-based assemblies.
Authors
Nonappa1,2 and Peter Engelhardt1
Affiliation
1Department of Applied Phyiscs, Aalto University School of Science, Espoo, Finland
2Department of Bioproducts and Biosystems, Aalto University School of Chemical Engineering, Espoo, Finland
Contact
Dr. Nonappa
Department of Applied Phyiscs
Aalto University School of Science
Espoo, Finland
nonappa@aalto.fi
More information on Electron Tomography
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References
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