Electron Microscopy and Diffraction of Wet Unstained and Unfixed Biological Objects

Parsons, Donald F.; Matricardi, V. R.; Moretz, R. C.; Turner, James N.

doi:10.1016/b978-0-12-005215-8.50012-7

Cited by 91 publications

(55 citation statements)

References 160 publications

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“…Such devices employ thin silicon nitride membranes to form a micro-scaled imaging chamber. Building upon the pioneering work of Parsons and colleagues [7][8][9][10] we provide a useful strategy to image individual viral assemblies in solution. The protocol described here explains methodologies that are compatible with single particle image processing routines and 3D reconstruction calculations.…”

Section: Discussionmentioning

confidence: 99%

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<em>In situ</em> TEM of Biological Assemblies in Liquid

Dukes

Gilmore

Tanner

et al. 2013

JoVE

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Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments-viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment. Video LinkThe video component of this article can be found at

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Section: Discussionmentioning

confidence: 99%

“…Electron diffraction patterns of unstained catalase crystals were successfully recorded to a resolution of 3 Å in a hydrated state [7][8] . In addition, phase-separated lipid domains could be examined in hydrated membranes of human erythrocytes [9][10] .…”

Section: Introductionmentioning

confidence: 99%

<em>In situ</em> TEM of Biological Assemblies in Liquid

Dukes

Gilmore

Tanner

et al. 2013

JoVE

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“…11a), which was few tens of microns away from the membrane. The O 1s spectra were deconvoluted using three components, assigned to OH, H 2 O (L) and H 2 O (G), (here L and G stand for liquid and vapor, correspondingly) based on prior research [86][87]. The presence of molecular water and hydroxyl adsorbed species beyond the orifice indicate that some water was trapped under the graphene during the wet transfer procedure or diffused from the orifice [88,23].…”

Section: First Ap-spem Experiments Through 2d Membranesmentioning

confidence: 99%

Recent Approaches for Bridging the Pressure Gap in Photoelectron Microspectroscopy

et al. 2016

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Ambient-pressure photoelectron spectroscopy (APPES) and microscopy are at the frontier of modern chemical analysis at liquid-gas, solid-liquid and solid-gas interfaces, bridging science and engineering of functional materials. Complementing the current state-of-the art of the instruments, we survey in this short review several alternative APPES approaches, developed recently in the scanning photoelectron microscope (SPEM) at the Elettra laboratory. In particular, we report on experimental setups for dynamic near-ambient pressure environment, using pulsed-gas injection in the vicinity of samples or reaction cells with very small apertures, allowing for experiments without introducing additional differential pumping stages. The major part of the review is dedicated to the construction and performance of novel environmental cells using ultrathin electron-transparent but molecularly impermeable membranes to isolate the gas or liquid ambient from the electron detector operating in ultra-high vacuum (UHV). We demonstrate that two dimensional materials, such as graphene and derivatives, are mechanically robust to withstand atmospheric - UHV pressure differences and are sufficiently transparent for the photoelectrons emitted from samples immersed in the liquid or gaseous media. There are many unique opportunities for APPES using X-rays over a wide energy range. We show representative results that illustrate the potential of these ‘ambient-pressure’ approaches. Combined with the ca 100 nm lateral resolution of SPEM, they can overcome the pressure gap challenges and address the evolution of chemical composition and electronic structure at surface and interfaces under realistic operation conditions with unprecedented lateral and spectral resolution.

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“…This phenomenon occurs by ionization of the gases inside the specimen chamber and nowadays much work has been done on it. The gas itself can be employed as a detector in the microscope system which is another advantage for ESEM (Parsons et al, 1974;Moncrieff et al, 1978;Crawford, 1979;DaniJatos, 1983DaniJatos, , 1986DaniJatos, , 1990Uwins, 1994). Study of the microstructure of goat SM muscle was carried out by Environmental Scanning Electron Microscopy (Yarmand & Homayouni, 2009;.…”

Section: Wwwintechopencommentioning

confidence: 99%