Super-resolution vibrational imaging using expansion stimulated Raman scattering microscopy

Stimulated Raman scattering (SRS) microscopy is an emerging technology that provides high chemical specificity for endogenous biomolecules and can circumvent common constraints of fluorescence microscopy including limited capabilities to probe small biomolecules and difficulty resolving many colors simultaneously due to spectral overlap. However, the resolution of SRS microscopy remains governed by the diffraction limit. To overcome this, we describe a new technique called Molecule Anchorable Gel-enabled Nanoscale Imaging of Fluorescence and stImulatEd Raman Scattering microscopy (MAGNIFIERS), that integrates SRS microscopy with expansion microscopy (ExM). ExM is a powerful strategy providing significant improvement in imaging resolution by physical magnification of hydrogel-embedded preserved biological specimens. MAGNIFIERS offers chemical-specific nanoscale imaging with sub-50 nm resolution and has scalable multiplexity when combined with multiplex Raman probes and fluorescent labels. We used MAGNIFIERS to visualize nanoscale features in a label-free manner with C-H vibration of proteins, lipids and DNA in a broad range of biological specimens, from mouse brain, liver and kidney to human lung organoid. In addition, we applied MAGNIFIERS to track nanoscale features of protein synthesis in protein aggregates using metabolic labeling of small metabolites. Finally, we used MAGNIFIERS to demonstrate 8-color nanoscale imaging in an expanded mouse brain section. Overall, MAGNIFIERS is a valuable platform for super-resolution label-free chemical imaging, high-resolution metabolic imaging, and highly multiplexed nanoscale imaging, thus bringing SRS to nanoscopy.

Standardizing Spatial Reconstruction Parameters for the Atom Probe Analysis of Common Minerals

Well-defined reconstruction parameters are essential to quantify the size, shape, and distribution of nanoscale features in atom probe tomography (APT) datasets. However, the reconstruction parameters of many minerals are difficult to estimate because intrinsic spatial markers, such as crystallographic planes, are not usually present within the datasets themselves. Using transmission and/or scanning electron microscopy imaging of needle-shaped specimens before and after atom probe analysis, we test various approaches to provide best-fit reconstruction parameters for voltage-based APT reconstructions. The results demonstrate that the length measurement of evaporated material, constrained by overlaying pre- and post-analysis images, yields more consistent reconstruction parameters than the measurement of final tip radius. Using this approach, we provide standardized parameters that may be used in APT reconstructions of 11 minerals. The adoption of standardized reconstruction parameters by the geoscience APT community will alleviate potential problems in the measurement of nanoscale features (e.g., clusters and interfaces) caused by the use of inappropriate parameters.

Dislocations as a Tool for Nanostructuring Advanced Materials

Dislocations present unique opportunities for nanostructuring advanced structural and functional materials due to the recent discoveries of linear complexions thermodynamically stable nanoscale features with unique chemistry and structure confined at dislocations. The formation of such features is driven by solute segregation near the dislocation core and results in the stabilization of dislocations, altering mechanical, thermodynamic, and transport properties of the final material. This perspective article gives an overview of the recent discoveries and predictions made by high-resolution experimental characterization techniques, as well as large-scale atomistic simulations in the newly emerging field of linear complexions.

Rapid, continuous projection multi-photon 3D printing enabled by spatiotemporal focusing of femtosecond pulses

There is demand for scaling up 3D printing throughput, especially for the multi-photon 3D printing process that provides sub-micrometer structuring capabilities required in diverse fields. In this work, high-speed projection multi-photon printing is combined with spatiotemporal focusing for fabrication of 3D structures in a rapid, layer-by-layer, and continuous manner. Spatiotemporal focusing confines printing to thin layers, thereby achieving print thicknesses on the micron and sub-micron scale. Through projection of dynamically varying patterns with no pause between patterns, a continuous fabrication process is established. A numerical model for computing spatiotemporal focusing and imaging is also presented which is verified by optical imaging and printing results. Complex 3D structures with smooth features are fabricated, with millimeter scale printing realized at a rate above 10−3 mm3 s−1. This method is further scalable, indicating its potential to make fabrications of 3D structures with micro/nanoscale features in a practical time scale a reality.

Rapid 3D-STORM imaging of diverse molecular targets in tissue

The precise organization of fine scale molecular architecture is critical for the nervous system and other biological functions and would benefit from nanoscopic imaging methods with improved accessibility, throughput, and native tissue compatibility. Here, we report RAIN-STORM, a rapid and scalable imaging approach that enables three-dimensional nanoscale target visualization for multiple subcellular and intracellular targets within tissue at depth. RAIN-STORM utilizes conventional tissue samples, readily available reagents in optimized formulas, requires no specialized sample handling, and is suitable for commercial instrumentation. To illustrate RAIN-STORM’s ability for quantitative high-resolution nanoscopic tissue imaging, we utilized the well-organized but structurally complex retina. We show that RAIN-STORM is rapid and versatile, enabling 3D nanoscopic imaging of over 20 distinct targets to reveal known and novel nanoscale features of synapses, neurons, glia, and vascular. Further, imaging parameters are compatible with a wide range of tissue sources and molecular targets across a spectrum of biological structures. Finally, we show that this method can be applied to clinically derived samples and reveal the nanoscale distribution of molecular targets within human samples. RAIN-STORM thus enables rapid 3D imaging for a range of molecules, paving the way for high throughput studies of nanoscopic molecular features in intact tissue from diverse sources.

Deconvolution of dissipative pathways for the interpretation of tapping-mode atomic force microscopy from phase-contrast

Phase-contrast in tapping-mode atomic force microscopy (TM-AFM) results from dynamic tip-surface interaction losses which allow soft and hard nanoscale features to be distinguished. So far, phase-contrast in TM-AFM has been interpreted using homogeneous Boltzmann-like loss distributions that ignore fluctuations. Here, we revisit the origin of phase-contrast in TM-AFM by considering the role of fluctuation-driven transitions and heterogeneous loss. At ultra-light tapping amplitudes <3 nm, a unique amplitude dependent two-stage distribution response is revealed, alluding to metastable viscous relaxations that originate from tapping-induced surface perturbations. The elastic and viscous coefficients are also quantitatively estimated from the resulting strain rate at the fixed tapping frequency. The transitional heterogeneous losses emerge as the dominant loss mechanism outweighing homogeneous losses at smaller amplitudes for a soft-material. Analogous fluctuation mediated phase-contrast is also apparent in contact resonance enhanced AFM-IR (infrared), showing promise in decoupling competing thermal loss mechanisms via radiative and non-radiative pathways. Understanding the loss pathways can provide insights on the bio-physical origins of heterogeneities in soft-bio-matter e.g., single cancer cell, tumors, and soft-tissues.

Nanoscale evidence of metamorphism – insights from natural and experimentally-treated zircon

&lt;p&gt;Nanoscale analyses of zircon have demonstrated that trace elements, including Pb, can be mobilized to discrete sites in radiation damaged zircon. Although several mechanisms for trace element mobility and segregation in zircon have been proposed, most of this work has been conducted on zircon grains with complex geologic histories, making it difficult to directly determine the mechanisms driving trace element mobility and segregation in zircon. To test among the existing hypotheses for mechanisms driving trace element mobility and segregation, we analyzed both untreated and experimentally heated (1450&amp;#176;C for 24h) Archean zircon using atom probe tomography and transmission electron microscopy (TEM). The sample has a simple, well-characterized thermal history, with no significant thermal events since original crystallization. Despite a high calculated radiation dose (&gt;4 x 10&lt;sup&gt;18&lt;/sup&gt; a/g), the untreated zircon does not contain anomalous nanoscale features. In contrast, the experimentally heated zircon contains abundant clusters of Y, Mg, Al, Pb + Yb that range from 5 nm to 25 nm in diameter with toroidal polyhedral morphologies. The &lt;sup&gt;207&lt;/sup&gt;Pb/&lt;sup&gt;206&lt;/sup&gt;Pb measured from Pb atoms located within these features is consistent with present-day segregation, thus confirming that these nanoscale features were produced by experimental heating in the laboratory. TEM analysis determined that the clusters are dislocation loops, and that cluster morphology is therefore crystallographically controlled. The largest loops are located in {100} and contain high concentrations of Mg and Al.&lt;/p&gt;&lt;p&gt;These experimentally induced, trace-element-enriched clusters are similar in size, morphology, composition, and crystallographic orientation to clusters observed in zircon affected by natural geologic processes (cf. Valley et al., 2015; Peterman et al., 2016). Although the calculated radiation doses for all analyzed grains are high, comparison of the nanoscale features indicates no apparent correlation between the radiation dose and the density or distribution of clusters. We also observe that trace-element-enriched clusters are conspicuously absent from zircon grains that lack younger igneous or metamorphic rims. These findings suggest that the pressure-temperature-time (P-T-t) history and the dT/dt significantly impact both the nanoscale redistribution of trace elements and the density of these features within zircon. Systematic evaluation of the composition and distribution of these features provides a framework for understanding the nanoscale record of metamorphism.&lt;/p&gt;&lt;p&gt;&amp;#160;&lt;/p&gt;&lt;p&gt;References:&lt;/p&gt;&lt;p&gt;Peterman, E.M., Reddy, S.M, Saxey, D.W., Snoeyenbos, D.R., Rickard, W.D.A., Fougerouse, D., and Kylander-Clark, A.R.C. (2016) Nanogeochronology of discordant zircon measured by atom probe microscopy of Pb-enriched dislocation loops. Science Advances, 2, e:1601218.&lt;/p&gt;&lt;p&gt;Valley, J.W., Reinhard, D.A., Cavosie, A.J., Ushikubo, T., Lawrence, D.F., Larson, D.J., Kelly, T.F., Snoeyenbos, DR., and Strickland, A. (2015) Nano-and micro-geochronology in Hadean and Archean zircons by atom-probe tomography and SIMS: New tools for old minerals. American Mineralogist, 100, 1355-1377.&lt;/p&gt;

Uncovering topographically hidden features in 2D MoSe2 with correlated potential and optical nanoprobes

Developing characterization strategies to better understand nanoscale features in two-dimensional nanomaterials is of crucial importance, as the properties of these materials are many times driven by nanoscale and microscale chemical and structural modifications within the material. For the case of large area monolayer MoSe2 flakes, kelvin probe force microscopy coupled with tip-enhanced photoluminescence was utilized to evaluate such features including internal grain boundaries, edge effects, bilayer contributions, and effects of oxidation/aging, many of which are invisible to topographical mapping. A reduction in surface potential due to n-type behavior was observed at the edge of the flakes as well as near grain boundaries. Potential phase mapping, which corresponds to the local dielectric constant, depicted local biexciton and trion states in optically-active regions of interest such as grain boundaries. Finally, nanoscale surface potential and photoluminescence mapping was performed at several stages of oxidation, revealing that various oxidative states can be evaluated during the aging process. Importantly, all of the characterization performed in this study was non-destructive and rapid, crucial for quality evaluation of an exciting class of two-dimensional nanomaterials.