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Electron Microscopy of Nucleic Acid Nanoparticles
Published in Peixuan Guo, Kirill A. Afonin, RNA Nanotechnology and Therapeutics, 2022
Damian Beasock, Kirill A. Afonin
Sample preparation depends on the technique being used. Most EM techniques require biological particles to be dehydrated, dispersed, and fixed onto a substrate before analysis [53]. TEM requires transmission of electron beam, and therefore NANPs need to be dispersed and fixed onto a TEM gird [54,55]. A TEM grid is a substrate designed to allow as many electrons as possible to pass through the sample. As nucleic acids are made of light elements (i.e. H, C, N, O, P), maximizing contrast between the substrate and the sample is important. This can be done by choosing a more electron transparent grid coating such as a thin low atomic number material [56]. Sample staining is also a helpful step to maximize contrast of nucleic acids. Heavy metal salts can be added to nucleic acids to bind high atomic number elements to certain sites [54,56–59]. Figure 23.1a and 23.1b shows an example of contrast between the sample and substrate. This is achieved when a strong difference in signal can be determined between the sample and the substrate. Figure 23.1c and 23.1d shows examples of samples prepared with positive and negative staining. Here, high atomic number elements such as tungsten, uranium, osmium, or lead bond to the NANP with positive staining or surround the NANP with negative staining. This is to enhance the contrast between the sample structure and the surroundings [60]. Instead of dehydrating biological samples, cryoEM sample preparation requires vitrification of the sample in amorphous ice. This helps preserve the in solution structure of the sample during visualization [61]. Proper sample preparation is meant to enhance quality and contrast of images to reveal as much about the NANP structure as possible.
Theoretical analyses of pressure induced glass transition in water: Signatures of surprising diffusion-entropy scaling across the transition
Published in Molecular Physics, 2021
Saumyak Mukherjee, Biman Bagchi
Glassy phases of water and their preparation techniques have fascinated researchers, both experimentalists and theoreticians, over many decades [1–10]. Amorphous ice phase is predicted to be the major state of water found in extra-terrestrial space. The low-temperature physics of water discusses a number of amorphous states, besides a complex array of crystalline phases [8,11–17]. Water has a rich and complicated phase diagram with 19 experimentally observed crystalline polymorphs (and several others observed in computer simulations) [18–22]. Cooling liquid water beyond its freezing temperature (273 K) is possible till ∼230 K, after which spontaneous crystallisation sets in. Several simulation studies propose that in this supercooled state, liquid water may have two distinct phases, namely high (HDL) and low (LDL) density liquid, with a critical point [5,9,14,15,23–26]. Further below in the temperature scale (beyond 130 K) glassy states including low (LDA), high (HDA) and very high (VHDA) density amorphous ice forms are found [5–7,10]. The methods of preparation of these phases (sudden temperature quenching, surface deposition of vapour, etc.), and the mutual transitions between them have fascinated researchers over a long time [5,8].
Structural differences between unannealed and expanded high-density amorphous ice based on isotope substitution neutron diffraction
Published in Molecular Physics, 2019
Katrin Amann-Winkel, Daniel T. Bowron, Thomas Loerting
We here compare isotope substitution neutron diffraction data for several sub-states of high-density amorphous ice (HDA). Specifically, we study the historically most studied form as prepared following the protocol of pressure-induced amorphization of ice Ih at 77 K [30]. This so-called unannealed HDA (uHDA) is compared with expanded forms of HDA (eHDA), namely two different types of eHDA – one that is decompressed to 0.20 GPa and one that is decompressed to 0.07 GPa at 140 K. These sub-states of HDA are also compared with very-high density amorphous ice (VHDA), which does not belong to the HDA family of states [6].