Roundabouts
John R. Helliwell in The Whats of a Scientific Life, 2019
Are there other options rather than repeat the whole circle of experiments again? One can try a method altogether different than X-ray crystal structure analysis. A method that has improved a lot in the last ten years or so in my field of structural chemistry and biology is that of cryo electron microscopy. In this case, a crystal is not needed and instead multiple images of a single molecular complex are studied directly. This new method has allowed the breaking out of going round in circles for those impossible-to-crystallise cases. Why were they impossible to crystallise? This could be for a variety of reasons. Firstly, although a homogenously pure sample of these molecules could be prepared, they can each be too flexible to crystallise in a simple, repeating, 3D array. Secondly, it may not have been possible to prepare a pure enough sample for crystallisation. So important is the improved method of cryo electron microscopy for the field of structural biochemistry and structural biology that its lead developers shared the Nobel Prize for Chemistry in 2017: Jacques Dubochet, Joachim Frank and Richard Henderson [2].
Introduction to Cells, DNA, and Viruses
Patricia G. Melloy in Viruses and Society, 2023
Finally, virologists who observe viral particles in tissues might turn to microscopy techniques such as electron microscopy and fluorescence microscopy to study viral protein structure and their interaction with cells. Because of the high magnification and resolution capability of electron microscopy, this microscopy technique has historically been used most often to visualize viruses (Figure 1.5) (Alberts et al. 2019; Lostroh 2019). The techniques of atomic force microscopy and a specialized type of electron microscopy called cryo-electron microscopy are often used as well. Superresolution microscopy has increased the capabilities of light microscopy as well (Nobel Prize Outreach AB 2014). However, cellular changes resulting from viral infection can be observed using conventional light microscopy and have been used even before the viral particles themselves could be visualized (Lostroh 2019).
Instrumentation
Clive R. Bagshaw in Biomolecular Kinetics, 2017
Electron microscopy has long been used to study the gross shape of protein and DNA molecules. The method has taken on increasing importance when applied to unstained hydrated samples under cryo-conditions, where domains and secondary structure and even side chains have been resolved [583,584]. Electron microscopy readily resolves single molecules, although this method has generally not been considered as a “single-molecule” kinetic technique because individual molecules cannot be followed in real time once they are fixed or frozen. However, progress has been made in real-time imaging of wet samples using a thin environmental chamber to isolate the sample from the vacuum of the microscope [585]. Using gold-labeled antibodies as a probe, images have been acquired with 0.1 s time resolution [586,587]. To follow reactions discontinuously using conventional electron microscopy, reactants are rapidly mixed in solution and then rapidly quenched by freezing, akin to that used for EPR spectroscopy (see Section 7.6.5). Devices have been constructed to spray a grid with the reactants and quickly plunge the grid in to liquid ethane to stop the reaction [583,588–591]. The sample is then examined in the frozen state by cryo-electron microscopy methods. The method has a dead time of about 5 ms. Because molecules are viewed from a range of orientations, individual molecules are classified according to their orientation before averaging with similar molecules. This approach has the power to resolve different conformational states within the same ensemble sample, which may indicate dynamics. However, kinetic methods are required to explore the interconversion rates [583].
Electron microscopy overview of SARS-COV2 and its clinical impact
Published in Ultrastructural Pathology, 2022
Soheir Saiid Mansy, Mona Mahmoud AbouSamra
Many techniques, including NMR spectroscopy, X-ray solution scattering, neutron diffraction, various spectroscopic techniques, and X-ray crystallography, have been used to determine the shape and structure of biological molecules. Recently, cryo-electron microscopy has become the most effective tool in structural biology after the technical development of its resolutions, which permits the identification of the biomolecular structure in its natural state.59 Cryo-EM has an advantage over X-ray crystallography, and is the most effective tool in analyzing macromolecules during the last few years. Cryo-EM reveals structures in fast-frozen non-crystalline biological samples that are closer to their natural state at an atomic level. In addition, it requires much smaller macromolecule samples to work with, unlike X-ray crystallography, which needs large pieces of materials to optimize the crystallization conditions.59 Hence, cryo-EM has become the tool of choice for determining the structure of macromolecular complexes, especially supra-assemblies that are difficult to prepare in large quantities or virtually inaccessible to crystallize.59,61,62 Identifying the structural biology of viral protein complexes at molecular resolution is important for designing small drug molecules to bind and impair their function.32
Use of electron microscopy to study platelets and thrombi
Published in Platelets, 2020
Maurizio Tomaiuolo, Rustem I. Litvinov, John W. Weisel, Timothy J. Stalker
Cryogenic electron microscopy (Cryo-EM) is a relatively new and rapidly developing type of TEM of hydrated biological samples that are instantaneously cooled to cryogenic temperatures (liquid ethane) without fixation and dehydration. The cooling procedure is so fast that water molecules do not form crystals that damage the structures but acquire a state of amorphous or vitreous ice that preserves the native biological organization of macromolecules, supramolecular structures, and cellular organelles. The cryogenically prepared samples can be analyzed using TEM, including tomography that involves acquisition of multiple images of the tilted specimen followed by 3D reconstruction [99]. Cryo-electron tomography (cryo-ET) of platelets combined with immunogold labeling was used to characterize the 3-dimensional organization of the platelet open canalicular system and its relationship to the dense tubular system, as well as to define α-granule subtypes [100]. Cryo-ET of intact platelets allowed visualization of the cytoskeletal architecture of spread platelets on extracellular matrix correlated with stiffness maps of the platelets determined with atomic force microscopy [101]. Using cryo-ET to examine frozen-hydrated platelets from patients with ovarian cancer revealed significant morphological differences between the cancer and control platelets, including disruption of the microtubule marginal band as well as differences in mitochondria and other subcellular structures [102]. These and other papers suggest the potential of cryo-EM as a powerful technology to study the ultrastructure of platelets in their native hydrated state at nanometer to subnanometer resolution [103].
A beacon for broader impact: illuminating science
Published in Journal of Visual Communication in Medicine, 2019
Our time is one of rapid rate of change, especially in science and technology; it is known as the ‘The Digital Age’, ‘The Century of Biology’, and ‘The Information Age’. With new advances in imaging, like cryo-electron microscopy, we have insight into biological structures like never before. With technologies in science, such as CRISPR-Cas9 genome editing, a system adept at knocking out mutations, researchers have opened a new gamut of potential applications in genetics.
Related Knowledge Centers
- Cryogenics
- Electron Diffraction
- Ethane
- Liquid Helium
- Propane
- Transmission Electron Microscopy
- Valine
- Vitrification
- X-Ray Crystallography
- Liquid Nitrogen