Exploring the effects of graphene and temperature in reducing electron beam damage: A TEM and electron diffraction-based quantitative study on Lead Phthalocyanine (PbPc) crystals

Jain, Noopur; Hao, Yansong; Parekh, Urvi; Kaltenegger, Martin; Pedrazo‐Tardajos, Adrián; Lazzaroni, Roberto; Resel, Roland; Geerts, Yves; Bals, Sara; Aert, Sandra Van

doi:10.1016/j.micron.2023.103444

Cited by 4 publications

(10 citation statements)

References 34 publications

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“…Using the graphene encapsulation method developed previously ( 24, 25 ), it is possible to confine biological samples within graphene encapsulated aqueous environments in standard Au-flat and Au Quantifoil grids (Fig.1A-D). A single-layer graphene grid was submerged in a container with a biological buffer of interest and the sample was pipetted over it.…”

Section: Resultsmentioning

confidence: 99%

“…The top graphene layer for encapsulation was fabricated using a polymer-free transfer method ( 55 ), which provides a flat top surface to the hydrated environment which then adapts to the morphology of the target. The main steps are described in detail in refs ( 56 ) and ( 25 ) to prepare the base TEM grid and the creation of the encapsulated environments respectively ( 57 ).…”

Section: Methodsmentioning

confidence: 99%

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Liquid phase electron microscopy of bacterial ultrastructure

Caffrey,

Pedrazo-Tardajos,

Liberti

et al. 2024

Preprint

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Recent advances in liquid phase scanning transmission electron microscopy (LP-STEM) have enabled the study of dynamic biological processes at nanometre resolutions, paving the way for live-cell imaging using electron microscopy. However, this technique is often hampered by the inherent thickness of whole cell samples and damage from electron beam irradiation. These restrictions degrade image quality and resolution, impeding biological interpretation. Here we detail the use of graphene encapsulation, STEM, and energy-dispersive X-ray (EDX) spectroscopy methods to mitigate these issues, providing unprecedented levels of intracellular detail in aqueous specimens. We demonstrate the feasibility of our approach using a radiation resistant, gram-positive bacterium, Deinococcus radiodurans, chosen specifically for its tolerance to the highly oxidative environments created by electron irradiation. This work shows the potential of LP-STEM to examine internal cellular structures in thick biological samples and identify key ultrastructure in these samples using a variety of imaging techniques.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Liquid phase electron microscopy of bacterial ultrastructure

Caffrey,

Pedrazo-Tardajos,

Liberti

et al. 2024

Preprint

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“…Furthermore, as graphene acts as a radical scavenger when the electron beam interacts with the liquid, the maximum electron dose (e-dose) that particles can sustain before losing their 3D order is higher. A higher e-dose can thus be used to achieve a higher signal-to-noise ratio during data collection. ,, …”

Section: Resultsmentioning

confidence: 99%

“…A higher e-dose can thus be used to achieve a higher signal-to-noise ratio during data collection. 17 , 18 , 25 …”

Section: Resultsmentioning

confidence: 99%

“…A higher e-dose can thus be used to achieve a higher signal-to-noise ratio during data collection. 17,18,25 Another consideration in LPEM sample preparation is the size and shape of the sample. Introducing particles with an extended dimension, such as platelets or needles, into a prefabricated liquid cell can be challenging or even impossible.…”

Section: Preparation Of Liquid Cell With Lysozyme Nanocrystalsmentioning

confidence: 99%

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High-Resolution Electron Diffraction of Hydrated Protein Crystals at Room Temperature

Plana-Ruiz,

Gómez-Pérez,

Budayova-Spano

et al. 2023

ACS Nano

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Structural characterization is crucial to understanding protein function. Compared with X-ray diffraction methods, electron crystallography can be performed on nanometer-sized crystals and can provide additional information from the resulting Coulomb potential map. Whereas electron crystallography has successfully resolved three-dimensional structures of vitrified protein crystals, its widespread use as a structural biology tool has been limited. One main reason is the fragility of such crystals. Protein crystals can be easily damaged by mechanical stress, change in temperature, or buffer conditions as well as by electron irradiation. This work demonstrates a methodology to preserve these nanocrystals in their natural environment at room temperature for electron diffraction experiments as an alternative to existing cryogenic techniques. Lysozyme crystals in their crystallization solution are hermetically sealed via graphene-coated grids, and their radiation damage is minimized by employing a low-dose data collection strategy in combination with a hybrid-pixel direct electron detector. Diffraction patterns with reflections of up to 3 Å are obtained and successfully indexed using a template-matching algorithm. These results demonstrate the feasibility of in situ protein electron diffraction. The method described will also be applicable to structural studies of hydrated nanocrystals important in many research and technological developments.

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