Identifying Nanotoxicity at the Cellular Level Using Electron Microscopy
Suresh C. Pillai, Yvonne Lang in Toxicity of Nanomaterials, 2019
Conventionally it is regarded that there are two main types or modes of electron microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). There have been many adaptations of these techniques, the base microscopes and their associated specimen preparations which include low temperature and cryo techniques. To ensure that the specimen is able to withstand the electron beam, various preparatory techniques (Figure 7.1) must be carried out and will be discussed in greater detail in Section 7.3. When gathering three-dimensional (3D) information about a specimen, the microscopist generally employs the SEM, where it is used to visualise the surface topography of a specimen. TEM images are generally two dimensional (2D) in nature but in recent years and with advances in technology, computational power, and software packages a series of 2D TEM images can be collected via electron tomography (ET) and reconstructed to create a 3D model of the structure of interest.
Application of Nonlinear Microscopy in Life Sciences
Lingyan Shi, Robert R. Alfano in Deep Imaging in Tissue and Biomedical Materials, 2017
The missing cone problem is a limitation common to many imaging methods, where a 3D object is projected on a 2D detector and geometrical constraints forbid observing the sample from some angles. This includes tilt-series transmission electron tomography, some forms of x-ray (computer) tomography, and also widefield fluorescence microscopy. It can be fully appreciated when the image formation process is formulated in spatial frequency domain [28], where the extent of the optical transfer function (OTF, a Fourier transform of the point spread function—PSF) drops to zero for angles near the optical axis. A real-life demonstration of this limitation is when one attempts to image a perfectly homogeneous fluorescent horizontal layer of infinite extent (much larger than the field of view, with perfectly even illumination): one cannot determine whether the layer is in focus, as the intensity of the (otherwise perfectly uniform) image of the layer does not change appreciably with defocus.
Pathogenesis
Marie Studahl, Paola Cinque, Tomas Bergström in Herpes Simplex Viruses, 2017
HSV-1 is a typical herpes virus consisting of a double-stranded DNA constituting of an electron-dense core within the icosahedral nucleocapsid built up by 162 capsomers. The nucleocapsid is surrounded by the adherent tegument, which in turn is tightly connected to the envelope. The structure of the enveloped particle was recently resolved by cryo-electron tomography (Fig. 1), where the tegument appeared to be asymmetric due to eccentric positioning of the nucleocapsid and displayed a partly filamentous, actin-like structure (1). Moreover, protruding envelope glycoproteins are of several morphological types, and these spikes tended to be nonrandomly clustered, which could be of possible functional importance during viral entry. Although HSV-2 virions seem to be more fragile and therefore less studied, the similarity of viral genes and their organization and expression between the two subtypes (see chap. 1) argues for a similarity in structure also.
Use of electron microscopy to study platelets and thrombi
Published in Platelets, 2020
Maurizio Tomaiuolo, Rustem I. Litvinov, John W. Weisel, Timothy J. Stalker
A major limitation of conventional TEM is the lack of 3-dimensional information when imaging thin sections. Besides advances in tissue preservation protocols, advances in microscope technology coupled with computational tools and image processing techniques now provide for 3-dimensional reconstruction of images obtained from serial tissue sections. Moreover, electron tomography (ET) is an approach whereby a single relatively thick tissue section is imaged by TEM at multiple angles (see Engberts et al for a methodological review [97]). The resulting images may then be computationally reconstructed into a 3-dimensional volume. ET is characterized by outstanding axial resolution (as low as 2 nm), making it particularly useful for imaging of fine membranous structures, including membrane pores, and individual macromolecules can even be localized and identified. The thickness of the tissue section to be imaged is limited to 200–300 nm. ET may be coupled with cryopreservation of cells (cryo-ET) to achieve outstanding resolution of structures in near-native states, which is discussed in the next section.
Release of α-granule contents during platelet activation
Published in Platelets, 2022
Classically depicted as spherical organelles with a peripheral membrane and central contents of soluble proteins. More recently, three-dimensional reconstruction of electron tomography identified rare morphologically distinct subpopulations, including tubular α-granules [12]. Consistent with the apparent rarity of granule subpopulations in human platelets, three-dimensional analysis of sequential block face electron microscope images shows variation in α-granule size and shape follows that of a normal distribution with most α-granules having a simple ovoid shape [7]. Whether rare shapes represent dynamic, functional intermediates or stable minor subpopulations remains currently a question of interpretation [8,13]. Tubular α-granules may form upon activation or be endocytic fusion intermediates. In mouse platelets, it has been suggested that variations in granule length could be due to granule-granule fusion [11]. For now, it is unclear how dynamic α-granules may be in resting human or mouse platelets.
Rigid monoclonal antibodies improve detection of SARS-CoV-2 nucleocapsid protein
Published in mAbs, 2021
Curtis D. Hodge, Daniel. J. Rosenberg, Patricia Grob, Mateusz Wilamowski, Andrzej Joachimiak, Greg L. Hura, Michal Hammel
IgG flexibility, its importance in improving mAb recognition, and its influence on agglutination have remained uncharacterized. Although there have been several attempts by cryo-electron tomography25–28 and negative stain (NS) electron tomography,29 large-scale flexibility measurements are often not amenable to single-particle techniques. In contrast, the resolution of small-angle X-ray scattering (SAXS) is sufficient, especially when atomic structures of individual components are available, to determine the conformational variability of the antigen-binding fragments (Fabs) in various antibodies,30 including complexes with antigens or Fc-gamma receptors (FcγRs).31,32 A previous study showed that the Fabs’ conformational flexibility is derived from the inherent plasticity of the Fc-hinge regions in solution.33 Rigidity of the hinges inversely correlates with, and can modulate mAb agonistic potency,34,35 and this highlights the importance of newer strategies to modulate antibody-agglutination.36
Related Knowledge Centers
- Macromolecule
- Peptide
- Protein
- Protein Tertiary Structure
- Transmission Electron Microscopy
- Tomography
- Cell
- High-Resolution Transmission Electron Microscopy
- Scanning Transmission Electron Microscopy
- Crowther Criterion