Three-Dimensional Scanning Transmission Electron Microscopy of Biological Specimens

Abstract

A three-dimensional (3D) reconstruction of the cytoskeleton and a clathrin-coated pit in mammalian cells has been achieved from a focal-series of images recorded in an aberration-corrected scanning transmission electron microscope (STEM). The specimen was a metallic replica of the biological structure comprising Pt nanoparticles 2–3 nm in diameter, with a high stability under electron beam radiation. The 3D dataset was processed by an automated deconvolution procedure. The lateral resolution was 1.1 nm, set by pixel size. Particles differing by only 10 nm in vertical position were identified as separate objects with greater than 20% dip in contrast between them. We refer to this value as the axial resolution of the deconvolution or reconstruction, the ability to recognize two objects, which were unresolved in the original dataset. The resolution of the reconstruction is comparable to that achieved by tilt-series transmission electron microscopy. However, the focal-series method does not require mechanical tilting and is therefore much faster. 3D STEM images were also recorded of the Golgi ribbon in conventional thin sections containing 3T3 cells with a comparable axial resolution in the deconvolved dataset.

Electron tomography simulator with realistic 3D phantom for evaluation of acquisition, alignment and reconstruction methods

“…Sample warping, also termed sample distortion, is attributed to strains created by inhomogeneous mass loss (de Jonge et al, 2010; Duan et al, 2014; Suzuki, 2002). The fraction of distortion may be modeled by a convergence to a fixed constant σ ∞ as the number of exposures increases.…”

Three-dimensional imaging of human stem cells using soft X-ray tomography

“…On the other hand, TEM provides high-resolution two-dimensional information, but is limited by the harsh fixation and sectioning methods necessary and incompatibility with specific antibody staining. In addition, while it is possible to reconstruct three-dimensional tomographic images using two-dimensional electron tomography [14,15], this method is very time consuming and suffers as tissue is lost in the sectioning process and use of harsh fixatives and contrast agents [16].…”

Three-dimensional locations of gold-labeled proteins in a whole mount eukaryotic cell obtained with 3nm precision using aberration-corrected scanning transmission electron microscopy

“…Recently, our group introduced the use of aberration-corrected STEM to obtain 3D reconstructions of biological structures with nanometer resolution (de Jonge et al, 2010a; De Jonge et al, 2007b). Lateral scanning of the sample over consecutive focal depths avoids the difficulties associated with imaging a specimen at high tilt angles (Koning and Koster, 2009).…”