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1. Cu2+-based distance measurements by pulsed EPR provide distance constraints for DNA backbone conformations in solution

   https://doi.org/10.1093/nar/gkaa133  

   Ghosh, Shreya ; Lawless, Matthew J ; Brubaker, Hanna J ; Singewald, Kevin ; Kurpiewski, Michael R ; Jen-Jacobson, Linda ; Saxena, Sunil ( February 2020 , Nucleic Acids Research)

   Abstract Electron paramagnetic resonance (EPR) has become an important tool to probe conformational changes in nucleic acids. An array of EPR labels for nucleic acids are available, but they often come at the cost of long tethers, are dependent on the presence of a particular nucleotide or can be placed only at the termini. Site directed incorporation of Cu2+-chelated to a ligand, 2,2′dipicolylamine (DPA) is potentially an attractive strategy for site-specific, nucleotide independent Cu2+-labelling in DNA. To fully understand the potential of this label, we undertook a systematic and detailed analysis of the Cu2+-DPA motif using EPR and molecular dynamics (MD) simulations. We used continuous wave EPR experiments to characterize Cu2+ binding to DPA as well as optimize Cu2+ loading conditions. We performed double electron-electron resonance (DEER) experiments at two frequencies to elucidate orientational selectivity effects. Furthermore, comparison of DEER and MD simulated distance distributions reveal a remarkable agreement in the most probable distances. The results illustrate the efficacy of the Cu2+-DPA in reporting on DNA backbone conformations for sufficiently long base pair separations. This labelling strategy can serve as an important tool for probing conformational changes in DNA upon interaction with other macromolecules. 

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2. Utilization of Remote Access Electron Microscopes to Enhance Technology Education and Foster STEM Interest in Preteen Students

   https://doi.org/10.1007/s11165-020-09964-4  

   Wolf, Vanessa ; Hsiao, Valerie ; Rodriguez, Brandon ; Min, Ashley ; Mayorga, Jill ; Ashcroft, Jared ( November 2020 , Research in Science Education)

   null (Ed.)

   Remote access technology in STEM education fills dual roles as an educational tool to deliver science education (Educational Technology) and as a means to teach about technology itself (Technology Education). A five-lesson sequence was introduced to 11 and 12-year-old students at an urban school. The lesson sequences were inquiry-based, hands-on, and utilized active learning pedagogies, which have been implemented in STEM classrooms worldwide. Each lesson employed a scanning electron microscope (SEM) and energy dispersive spectroscopy (EDS) accessed remotely. Students were assessed using multiple-choice questions to ascertain (1) technology education learning gains: did students gain an understanding of how electron microscopes work? and (2) educational technology learning gains: did students gain a better understanding of lesson content through use of the electron microscope? Likert-item surveys were developed, distributed, and analyzed to established how remote access technology affected student attitudes toward science, college, and technology. Participating students had a positive increase in attitudes toward scientific technology by engaging in the lesson sequences, reported positive attitudes toward remote access experiences, and exhibited learning gains in the science behind the SEM technology they accessed remotely. These findings suggest that remote experiences are a strong form of technology education, but also that future research could explore ways to strengthen remote access as an educational technology (a tool to deliver lesson content), such as one-on-one engagement. This study promotes future research into inquiry-based, hands-on, integrated lessons approach that utilize educational technology learning through remote instruments as a pedagogy to increase students’ engagement with and learning of the T in STEM. 

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3. Gd 3+ –Gd 3+ distances exceeding 3 nm determined by very high frequency continuous wave electron paramagnetic resonance

   https://doi.org/10.1039/c6cp07119h  

   Clayton, Jessica A. ; Qi, Mian ; Godt, Adelheid ; Goldfarb, Daniella ; Han, Songi ; Sherwin, Mark S. ( January 2017 , Physical Chemistry Chemical Physics)

   Electron paramagnetic resonance spectroscopy in combination with site-directed spin labeling is a very powerful tool for elucidating the structure and organization of biomolecules. Gd 3+ complexes have recently emerged as a new class of spin labels for distance determination by pulsed EPR spectroscopy at Q- and W-band. We present CW EPR measurements at 240 GHz (8.6 Tesla) on a series of Gd-rulers of the type Gd-PyMTA–spacer–Gd-PyMTA, with Gd–Gd distances ranging from 1.2 nm to 4.3 nm. CW EPR measurements of these Gd-rulers show that significant dipolar broadening of the central |−1/2〉 → |1/2〉 transition occurs at 30 K for Gd–Gd distances up to ∼3.4 nm with Gd-PyMTA as the spin label. This represents a significant extension for distances accessible by CW EPR, as nitroxide-based spin labels at X-band frequencies can typically only access distances up to ∼2 nm. We show that this broadening persists at biologically relevant temperatures above 200 K, and that this method is further extendable up to room temperature by immobilizing the sample in glassy trehalose. We show that the peak-to-peak broadening of the central transition follows the expected 1/ r 3 dependence for the electron–electron dipolar interaction, from cryogenic temperatures up to room temperature. A simple procedure for simulating the dependence of the lineshape on interspin distance is presented, in which the broadening of the central transition is modeled as an S = 1/2 spin whose CW EPR lineshape is broadened through electron–electron dipolar interactions with a neighboring S = 7/2 spin. 

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4. Realizing the Full Potential of Advanced Microscopy Approaches for Interrogating Plant-Microbe Interactions

   https://doi.org/10.1094/MPMI-10-22-0208-FI  

   Czymmek, Kirk J. ; Duncan, Keith E. ; Berg, Howard ( April 2023 , Molecular Plant-Microbe Interactions®)

   Microscopy has served as a fundamental tool for insight and discovery in plant-microbe interactions for centuries. From classical light and electron microscopy to corresponding specialized methods for sample preparation and cellular contrasting agents, these approaches have become routine components in the toolkit of plant and microbiology scientists alike to visualize, probe and understand the nature of host-microbe relationships. Over the last three decades, three-dimensional perspectives led by the development of electron tomography, and especially, confocal techniques continue to provide remarkable clarity and spatial detail of tissue and cellular phenomena. Confocal and electron microscopy provide novel revelations that are now commonplace in medium and large institutions. However, many other cutting-edge technologies and sample preparation workflows are relatively unexploited yet offer tremendous potential for unprecedented advancement in our understanding of the inner workings of pathogenic, beneficial, and symbiotic plant-microbe interactions. Here, we highlight key applications, benefits, and challenges of contemporary advanced imaging platforms for plant-microbe systems with special emphasis on several recently developed approaches, such as light-sheet, single molecule, super-resolution, and adaptive optics microscopy, as well as ambient and cryo-volume electron microscopy, X-ray microscopy, and cryo-electron tomography. Furthermore, the potential for complementary sample preparation methodologies, such as optical clearing, expansion microscopy, and multiplex imaging, will be reviewed. Our ultimate goal is to stimulate awareness of these powerful cutting-edge technologies and facilitate their appropriate application and adoption to solve important and unresolved biological questions in the field. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license . 

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5. Solving the iron quantification problem in low-kV EPMA: An essential step toward improved analytical spatial resolution in electron probe microanalysis—Olivines

   https://doi.org/10.2138/am-2019-6865  

   Moy, Aurélien ; Fournelle, John H. ; von der Handt, Anette ( August 2019 , American Mineralogist)

   Abstract The relatively recent entry of field emission electron microprobes into the field of microanalysis provides another tool for the study of small features of interest (e.g., mineral and melt inclusions, ex-solution lamellae, grain boundary phases, high-pressure experimental charges). However, the critical limitation for accurate quantitative analysis of these submicrometer- to micrometer-sized features is the relationship between electron beam potential and electron scattering within the sample. To achieve submicrometer analytical volumes from which X-rays are generated, the beam accelerating voltage must be reduced from 15–20 to ≤10 kV (often 5 to 7 kV) to reduce the electron interaction volume from ~3 to ~0.5 μm in common geological materials. At these low voltages, critical Kα X-ray lines of transition elements such as Fe are no longer generated, so L X-ray lines must be used. However, applying the necessary matrix corrections to these L lines is complicated by bonding and chemical peak shifts for soft X-ray transitions such as those producing the FeLα X-ray line. It is therefore extremely challenging to produce accurate values for Fe concentration with this approach. Two solutions have been suggested, both with limitations. We introduce here a new, simple, and accurate solution to this problem, using the common mineral olivine as an example. We also introduce, for the first time, olivine results from a new analytical device, the Extended Range Soft X-ray Emission Spectrometer. 

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