The electronic, optical, and mechanical properties of bilayer and trilayer graphene vary with their structure, including the stacking order and relative twist, providing novel ways to realize useful characteristics not available to single layer graphene. However, developing controlled growth of bilayer and trilayer graphene requires efficient large-scale characterization of multilayer graphene structures. Here, we use dark-field transmission electron microscopy for rapid and accurate determination of key structural parameters (twist angle, stacking order, and interlayer spacing) of few-layer CVD graphene. We image the long-range atomic registry for oriented bilayer and trilayer graphene, find that it conforms exclusively to either Bernal or rhombohedral stacking, and determine their relative abundances. In contrast, our data on twisted multilayers suggest the absence of such long-range atomic registry. The atomic registry and its absence are consistent with the two different strain-induced deformations we observe; by tilting the samples to break mirror symmetry, we find a high density of twinned domains in oriented multilayer graphene, where multiple domains of two different stacking configurations coexist, connected by discrete twin boundaries. In contrast, individual layers in twisted regions continuously stretch and shear independently, forming elaborate Moiré patterns. These results, and the twist angle distribution in our CVD graphene, can be understood in terms of an angle-dependent interlayer potential model.
Correlative microscopy, the integration of two or more microscopy techniques performed on the same sample, produces results that emphasize the strengths of each technique while offsetting their individual weaknesses. Light microscopy has historically been a central method in correlative microscopy due to its widespread availability, compatibility with hydrated and live biological samples, and excellent molecular specificity through fluorescence labeling. However, conventional light microscopy can only achieve a resolution of ∼300 nm, undercutting its advantages in correlations with higher-resolution methods. The rise of super-resolution microscopy (SRM) over the past decade has drastically improved the resolution of light microscopy to ∼10 nm, thus creating exciting new opportunities and challenges for correlative microscopy. Here we review how these challenges are addressed to effectively correlate SRM with other microscopy techniques, including light microscopy, electron microscopy, cryomicroscopy, atomic force microscopy, and various forms of spectroscopy. Though we emphasize biological studies, we also discuss the application of correlative SRM to materials characterization and single-molecule reactions. Finally, we point out current limitations and discuss possible future improvements and advances. We thus demonstrate how a correlative approach adds new dimensions of information and provides new opportunities in the fast-growing field of SRM.
The application of electron microscopy to hydrated biological samples has been limited by high-vacuum operating conditions. Traditional methods utilize harsh and laborious sample dehydration procedures, often leading to structural artefacts and creating difficulties for correlating results with high-resolution fluorescence microscopy. Here, we utilize graphene, a single-atom-thick carbon meshwork, as the thinnest possible impermeable and conductive membrane to protect animal cells from vacuum, thus enabling high-resolution electron microscopy of wet and untreated whole cells with exceptional ease. Our approach further allows for facile correlative super-resolution and electron microscopy of wet cells directly on the culturing substrate. In particular, individual cytoskeletal actin filaments are resolved in hydrated samples through electron microscopy and well correlated with super-resolution results.
When cells enter meiosis, their chromosomes reorganize as linear arrays of chromatin loops anchored to a central axis. Meiotic chromosome axes form a platform for the assembly of the synaptonemal complex (SC) and play central roles in other meiotic processes, including homologous pairing, recombination, and chromosome segregation. However, little is known about the 3D organization of components within the axes, which include cohesin complexes and additional meiosis-specific proteins. Here, we investigate the molecular organization of meiotic chromosome axes in Caenorhabditis elegans through STORM (stochastic optical reconstruction microscopy) and PALM (photo-activated localization microscopy) superresolution imaging of intact germ-line tissue. By tagging one axis protein (HIM-3) with a photoconvertible fluorescent protein, we established a spatial reference for other components, which were localized using antibodies against epitope tags inserted by CRISPR/Cas9 genome editing. Using 3D averaging, we determined the position of all known components within synapsed chromosome axes to high spatial precision in three dimensions. We find that meiosis-specific HORMA domain proteins span a gap between cohesin complexes and the central region of the SC, consistent with their essential roles in SC assembly. Our data further suggest that the two different meiotic cohesin complexes are distinctly arranged within the axes: Although cohesin complexes containing the kleisin REC-8 protrude above and below the plane defined by the SC, complexes containing COH-3 or -4 kleisins form a central core, which may physically separate sister chromatids. This organization may help to explain the role of the chromosome axes in promoting interhomolog repair of meiotic double-strand breaks by inhibiting intersister repair.
The importance of context in regulation of gene expression is now an accepted principle; yet the mechanism by which the microenvironment communicates with the nucleus and chromatin in healthy tissues is poorly understood. A functional role for nuclear and cytoskeletal architecture is suggested by the phenotypic differences observed between epithelial and mesenchymal cells. Capitalizing on recent advances in cryogenic techniques, volume electron microscopy and super-resolution light microscopy, we studied human mammary epithelial cells in three-dimensional (3D) cultures forming growtharrested acini. Intriguingly, we found deep nuclear invaginations and tunnels traversing the nucleus, encasing cytoskeletal actin and/or intermediate filaments, which connect to the outer nuclear envelope. The cytoskeleton is also connected both to other cells through desmosome adhesion complexes and to the extracellular matrix through hemidesmosomes. This finding supports a physical and/or mechanical link from the desmosomes and hemidesmosomes to the nucleus, which had previously been hypothesized but now is visualized for the first time. These unique structures, including the nuclear invaginations and the cytoskeletal connectivity to the cell nucleus, are consistent with a dynamic reciprocity between the nucleus and the outside of epithelial cells and tissues.
Cellular uptake of nanoparticles (NPs) depends on the nature of the nanobio system including the solid nanocomponents ( e. g., physicochemical properties of NPs), nanobio interfaces ( e. g., protein corona composition), and the cellular characteristics ( e. g., cell type). In this study, we document the role of sex in cellular uptake of NPs as an "overlooked" factor in nanobio interface investigations. We demonstrate that cell sex leads to differences in NP uptake between male and female human amniotic stem cells (hAMSCs), with greater uptake by female cells. hAMSCs are one of the earliest sources of somatic stem cells. The experiments were replicated with primary fibroblasts isolated from the salivary gland of adult male and female donors of similar ages, and again the extent of NP uptake was altered by cell sex. However, in contrast to hAMSCs, uptake was greater in male cells. We also found out that female versus male amniotic stem cells exhibited different responses to reprogramming into induced pluripotent stem cells (iPSCs) by the Yamanaka factors. Thus, future studies should consider the effect of sex on the nanobio interactions to optimize clinical translation of NPs and iPSC biology and to help researchers to better design and produce safe and efficient therapeutic sex-specific NPs.
The discovery and rise of graphene were historically enabled by its ∼10% optical contrast on specialized substrates like oxide-capped silicon. However, substantially lower contrast is obtained on transparent substrates. Moreover, it remains difficult to visualize nanoscale defects in graphene, including voids, cracks, wrinkles, and multilayers, on most device substrates. We report the use of interference reflection microscopy (IRM), a facile, label-free optical microscopy method originated in cell biology, to directly visualize graphene on transparent inorganic and polymer substrates at 30-40% image contrast per graphene layer. Our noninvasive approach overcomes typical challenges associated with transparent substrates, including insulating and rough surfaces, enables unambiguous identification of local graphene layer numbers and reveals nanoscale structures and defects with outstanding contrast and throughput. We thus demonstrate in situ monitoring of nanoscale defects in graphene, including the generation of nanocracks under uniaxial strain, at up to 4× video rate.
SUMMARY Ion channels control sperm navigation within the female reproductive tract and, thus, are critical for their ability to find and fertilize an egg. The flagellar calcium channel CatSper controls sperm hyperactivated motility and is dependent on an alkaline cytoplasmic pH. The latter is accomplished by either proton transporters or, in human sperm, via the voltage-gated proton channel Hv1. To provide concerted regulation, ion channels and their regulatory proteins must be compartmentalized. Here, we describe flagellar regulatory nanodomains comprised of Hv1, CatSper, and its regulatory protein ABHD2. Super-resolution microscopy revealed that Hv1 is distributed asymmetrically within bilateral longitudinal lines and that inhibition of this channel leads to a decrease in sperm rotation along the long axis. We suggest that specific distribution of flagellar nanodomains provides a structural basis for the selective activation of CatSper and subsequent flagellar rotation. The latter, together with hyperactivated motility, enhances the fertility of sperm.
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