Thickness measurements of graphene oxide flakes using atomic force microscopy: results of an international interlaboratory comparison.

Flake thickness is one of the defining properties of graphene-related 2D materials (GR2Ms), and therefore requires reliable, accurate, and reproducible measurements with well-understood uncertainties. This is needed regardless of the production method or manufacturer because it is important for all GR2M products to be globally comparable. An international interlaboratory comparison on thickness measurements of graphene oxide flakes using atomic force microscopy has been completed in technical working area 41 of versailles project on advanced materials and standards. Twelve laboratories participated in the comparison project, led by NIM, China, to improve the equivalence of thickness measurement for two-dimensional flakes. The measurement methods, uncertainty evaluation and a comparison of the results and analysis are reported in this manuscript. The data and results of this project will be directly used to support the development of an ISO standard.

Dysregulated HIC1 and RassF1A expression in vitro alters the cell cytoskeleton and exosomal Piwi-interacting RNA.

HIC1 and RassF1A methylation, which cause loss of gene function, are found in various cancers, including renal cell carcinoma (RCC), and could alter cell stiffness and the content of extracellular vesicles (EVs). These physiological changes may provide a tumoral survival advantage and thus could serve as cellular biomarkers for monitoring cell transformation, although direct associations between these changes and cell transformation remain to be established. As we found HIC1 and RassF1A methylation and expression changes in RCC samples, we examined the effects of gain and loss of HIC1 and RassF1A expression on cell DNA content, cytoskeletal structure, and Piwi-interacting RNA (piRNA) expression in EVs. We found HIC1 and RassF1A hypermethylation and abnormal expression in RCC patient samples was independent of the somatic mutations found in publicly available data. Cell stiffness was reduced in accordance with disrupted cytoskeleton conformation after knockdown of HIC1 or RassF1A. Gain or loss of HIC1 expression induced instability in genomic content, abnormal RassF1A expression disturbed cytoskeletal structure, and the abnormal expression of either gene altered piRNA content in EVs. These results suggest a causal relationship between abnormal tumor suppressor gene expression, cell stiffness, and piRNA expression.

Nanoscale Material Heterogeneity of Glowworm Capture Threads Revealed by AFM.

Adhesive materials used by many arthropods for biological functions incorporate sticky substances and a supporting material that operate synergistically by exploiting substrate attachment and energy dissipation. While there has been much focus on the composition and properties of the sticky glues of these bio-composites, less attention has been given to the materials that support them. In particular, as these materials are primarily responsible for dissipation during adhesive pull-off, little is known of the structures that give rise to functionality, especially at the nano-scale. In this study we used tapping mode atomic force microscopy (TM-AFM) to analyze unstretched and stretched glowworm (Arachnocampa tasmaniensis) capture threads and revealed nano-scale features corresponding to variation in surface structure and elastic modulus near the surface of the silk. Phase images demonstrated a high resolution of viscoelastic variation and revealed mostly globular and elongated features in the material. Increased vertical orientation of 11-15 nm wide fibrillar features was observed in stretched threads. Fast Fourier transform analysis of phase images confirmed these results. Relative viscoelastic properties were also highly variable at inter- and intra-individual levels. Results of this study demonstrate the practical usefulness of TM-AFM, especially phase angle imaging, in investigating the nano-scale structures that give rise to macro-scale function of soft and highly heterogeneous materials of both natural and synthetic origins.

HIC1 and RassF1A Methylation Attenuates Tubulin Expression and Cell Stiffness in Cancer.

Cell stiffness is a potential biomarker for monitoring cellular transformation, metastasis, and drug resistance development. Environmental factors relayed into the cell may result in formation of inheritable markers (e.g., DNA methylation), which provide selectable advantages (e.g., tumor development-favoring changes in cell stiffness). We previously demonstrated that targeted methylation of two tumor suppressor genes, hypermethylated in cancer 1 (HIC1) and Ras-association domain family member 1A (RassF1A), transformed mesenchymal stem cells (MSCs). Here, transformation-associated cytoskeleton and cell stiffness changes were evaluated. Atomic force microscopy (AFM) was used to detect cell stiffness, and immunostaining was used to measure cytoskeleton expression and distribution in cultured cells as well as in vivo. HIC1 and RassF1A methylation (me_HR)-transformed MSCs developed into tumors that clonally expanded in vivo. In me_HR-transformed MSCs, cell stiffness was lost, tubulin expression decreased, and F-actin was disorganized; DNA methylation inhibitor treatment suppressed their tumor progression, but did not fully restore their F-actin organization and stiffness. Thus, me_HR-induced cell transformation was accompanied by the loss of cellular stiffness, suggesting that somatic epigenetic changes provide inheritable selection markers during tumor propagation, but inhibition of oncogenic aberrant DNA methylation cannot restore cellular stiffness fully. Therefore, cell stiffness is a candidate biomarker for cells' physiological status.

Measurements of an Effective Longitudinal Coherence Length in Transmission Small Angle X-ray Scatterings.

The notion of an effective longitudinal coherence length with its value much greater than λ2/(2Δλ) has been adopted in small-angle X-ray scattering communities for years, where λ and Δλ denote the incident wavelength and its spread, respectively. Often the implications of the effective longitudinal coherence length do not even enter considerations in the designing and data treatment of small-angle scattering experiments. In this work, conventional transmission small-angle X-ray scattering (tSAXS) was performed to reveal a clear angular dependence on effective longitudinal coherence length. The measured values of effective longitudinal coherence length can be as high as one millimeter, whereas the value of calculated λ2/(2Δλ) is in nanometers.