Single-orientation Quantitative Susceptibility Mapping identifies the Ventral Intermediate Nucleus of the Thalamus

Introduction: The ventral intermediate nucleus (VIM) represents the primary target in the treatment of tremor. Accurate localization is extremely important given its proximity to other thalamic nuclei. We utilized single orientation quantitative susceptibility mapping (QSM) at 3T to directly visualize the VIM. Methods: Four adult volunteers, one adult cadaver, and an essential tremor patient were scanned on a 3T MRI using a multi-echo gradient echo sequence. QSM images were generated using the improved sparse linear equation and least-squares (iLSQR) algorithm. Two adult subjects underwent multiple head orientation imaging for multi-orientation QSM reconstruction. The VIM was prospectively identified with direct visualization as well as indirect landmark-based localization. Results: The bilateral VIM was consistently identified as a hypointense structure within the lateral thalamus, appearing similar on multi-orientation and single-orientation QSM, corresponding to the myelinated dentatorubrothalamic tract (DRTT). The indirect method resulted in a comparatively inferomedial localization, at times missing the VIM and DRTT. Conclusion: Single-orientation QSM offers a clinically feasible, non-invasive imaging-based approach to directly localize the VIM.

Angle change of the A-domain in a single SERCA1a molecule detected by defocused orientation imaging

The sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) transports Ca2+ ions across the membrane coupled with ATP hydrolysis. Crystal structures of ligand-stabilized molecules indicate that the movement of actuator (A) domain plays a crucial role in Ca2+ translocation. However, the actual structural movements during the transitions between intermediates remain uncertain, in particular, the structure of E2PCa2 has not been solved. Here, the angle of the A-domain was measured by defocused orientation imaging using isotropic total internal reflection fluorescence microscopy. A single SERCA1a molecule, labeled with fluorophore ReAsH on the A-domain in fixed orientation, was embedded in a nanodisc, and stabilized on Ni–NTA glass. Activation with ATP and Ca2+ caused angle changes of the fluorophore and therefore the A-domain, motions lost by inhibitor, thapsigargin. Our high-speed set-up captured the motion during EP isomerization, and suggests that the A-domain rapidly rotates back and forth from an E1PCa2 position to a position close to the E2P state. This is the first report of the detection in the movement of the A-domain as an angle change. Our method provides a powerful tool to investigate the conformational change of a membrane protein in real-time.

Using Macrotexture Map to Visualize Texture Heterogeneity in Polycrystalline Aggregate

A new tool, macrotexture map, was developed to represent and visualize texture heterogeneity in polycrystalline aggregate. This is a critical tool for microstructure representation, useful in risk analysis, performance simulation, and hotspot identification. In contrast to orientation imaging microscope (OIM) map where each color represents a crystal orientation, each color in this macrotexture map represents a texture. Different color represent different texture and similar texture shall have similar color. Macrotexture map provide a unique function to quantitatively evaluate texture heterogeneity of large components, leading to a first-hand understanding of property heterogeneity and anisotropy. For an experienced user, these maps serve the same purpose in identifying high risk locations in the investigated component as medical imaging maps do for diagnosis purpose. This method will also serve as a starting point in mesoscale simulation with meshing sensitivity based on the texture heterogeneity. It will provide a bridge between texture characterization and behavior simulation of components with texture heterogeneity. Macrotexture map will offer a linkage between crystal plasticity simulation in small length scale and finite element/difference simulation in large length scale.

Study of diffusionless and diffusional transformations using in situ cooling and heating techniques in a scanning electron microscope

In situ electron microscopy can be an effective tool to investigate the underlying science of many transformation mechanisms in materials science. Useful utilization of these experimentations will provide greater insight into many of the existing theories, as microstructural changes can be visualized in real time under some applied constraints. In this study, we have investigated two basic phase transformation phenomena: diffusionless and diffusional mechanisms with the help of in situ cooling and heating techniques in scanning electron microscope (SEM). In situ cooling experiments have been carried out on secondary hardening ultra-high-strength steels to understand the diffusionless transformation of austenite to martensite. Nucleation and growth of the martensites have been observed with cooling in different steps to −194°C. Details of the formation of different variants of martensites in steel were studied with the help of orientation imaging microscopy. Diffusional transformations were studied in terms of oxidation of pure copper in SEM using in situ heating technique. Different heating cycles were adopted for different samples by in situ heating to a maximum temperature of 950°C for the oxidation study. Nucleation of copper oxides and subsequent growth of the copper oxides at different temperatures were studied systematically. Raman spectroscopy and orientation imaging were done to confirm the formation of oxides and their orientations. The thermal cycling phenomenon was replicated inside SEM with heating and cooling and it has been demonstrated how the nature of copper and its oxides changes with the thermal cycle. This article is part of a discussion meeting issue ‘Dynamic in situ microscopy relating structure and function’.

Transmission Electron Microscopy of the Retina: A Method for Sample Preparation and Evaluation

Ultrastructural pathology is critical in the morphologic evaluation and characterization of subcellular structures in nonclinical toxicity and efficacy studies. In murine models of ophthalmologic disease, clinical examination is typically paired with other techniques like electroretinography (ERG) and/or optical coherence tomography (OCT) to more fully characterize a finding. High-quality transmission electron microscopy (TEM) can provide a critical, image-based link between these approaches, providing greater confidence in interpretation of ERG or OCT results. In addition to characterization of disease models, TEM can provide detailed visualization of retinal changes identified by clinical examination or light microscopy in nonclinical toxicity studies. The spherical shape of the eye presents unique challenges for trimming, orientation, imaging, and evaluation by TEM. The varied components of the eye require specialized approaches for embedding to facilitate successful sectioning. Controlling for the orientation of the retina is critical to consistent evaluation, driving the need for an improved method of embedding this unique and complex organ. The authors describe a method of sample preparation resulting in optimal orientation of the posterior aspect of murine eyes (rat and mouse) for TEM of the neural retina, Bruch’s membrane and/or choroid, with examples from mouse ophthalmic disease models.