AbstractScanning transmission electron microscopy (STEM) is widely used for imaging, diffraction, and spectroscopy of materials down to atomic resolution. Recent advances in detector technology and computational methods have enabled many experiments that record a full image of the STEM probe for many probe positions, either in diffraction space or real space. In this paper, we review the use of these four-dimensional STEM experiments for virtual diffraction imaging, phase, orientation and strain mapping, measurements of medium-range order, thickness and tilt of samples, and phase contrast imaging methods, including differential phase contrast, ptychography, and others.
Abstract
Electron staining is generally performed prior to observing organic materials via transmission electron microscopy (TEM) to enhance image contrast. However, electron staining can deteriorate organic materials. Here, we demonstrate electrostatic potential imaging of organic materials via differential phase contrast (DPC) scanning transmission electron microscopy (STEM) without electron staining. Electrostatic potential imaging drastically increases the contrast between different materials. Phase-separated structures in a poly (3-hexylthiophene) (P3HT) and [6,6]-phenyl-C61-butyric acid methyl ester (PCBM) blend that are impossible to observe using conventional STEM are clearly visualized. Furthermore, annealing behavior of the phase-separated structures is directly observed. The morphological transformations in the samples are consistent with their physical parameters, including their glass transition and melting temperatures. Our results indicate that electrostatic potential imaging is highly effective for observing organic materials.
Abstract
Background
The evolution of cartilage degeneration is still not fully understood, partly due to its thinness, low radio-opacity and therefore lack of adequately resolving imaging techniques. X-ray phase-contrast imaging (X-PCI) offers increased sensitivity with respect to standard radiography and CT allowing an enhanced visibility of adjoining, low density structures with an almost histological image resolution. This study examined the feasibility of X-PCI for high-resolution (sub-) micrometer analysis of different stages in tissue degeneration of human cartilage samples and compare it to histology and transmission electron microscopy.
Methods
Ten 10%-formalin preserved healthy and moderately degenerated osteochondral samples, post-mortem extracted from human knee joints, were examined using four different X-PCI tomographic set-ups using synchrotron radiation the European Synchrotron Radiation Facility (France) and the Swiss Light Source (Switzerland). Volumetric datasets were acquired with voxel sizes between 0.7 × 0.7 × 0.7 and 0.1 × 0.1 × 0.1 µm3. Data were reconstructed by a filtered back-projection algorithm, post-processed by ImageJ, the WEKA machine learning pixel classification tool and VGStudio max. For correlation, osteochondral samples were processed for histology and transmission electron microscopy.
Results
X-PCI provides a three-dimensional visualization of healthy and moderately degenerated cartilage samples down to a (sub-)cellular level with good correlation to histologic and transmission electron microscopy images. X-PCI is able to resolve the three layers and the architectural organization of cartilage including changes in chondrocyte cell morphology, chondrocyte subgroup distribution and (re-)organization as well as its subtle matrix structures.
Conclusions
X-PCI captures comprehensive cartilage tissue transformation in its environment and might serve as a tissue-preserving, staining-free and volumetric virtual histology tool for examining and chronicling cartilage behavior in basic research/laboratory experiments of cartilage disease evolution.
Phase Contrast Imaging of Weakly-Scattering Samples with Matched Illumination and Detector Interferometry–Scanning Transmission Electron Microscopy (MIDI–STEM)
