Microyielding of Core-Shell Crystal Dendrites in a Bulk-metallic-glass Matrix Composite

In-situ synchrotron x-ray experiments have been used to follow the evolution of the diffraction peaks for crystalline dendrites embedded in a bulk metallic glass matrix subjected to a compressive loading-unloading cycle. We observe irreversible diffraction-peak splitting even though the load does not go beyond half of the bulk yield strength. The chemical analysis coupled with the transmission electron microscopy mapping suggests that the observed peak splitting originates from the chemical heterogeneity between the core (major peak) and the stiffer shell (minor peak) of the dendrites. A molecular dynamics model has been developed to compare the hkl-dependent microyielding of the bulk metallic-glass matrix composite. The complementary diffraction measurements and the simulation results suggest that the interface, as Maxwell damper, between the amorphous matrix and the (211) crystalline planes relax under prolonged load that causes a delay in the reload curve which ultimately catches up with the original path

Expansion microscopy of C. elegans.

Funder: John DoerrFunder: The Open Philanthropy ProjectFunder: Lisa YangWe recently developed expansion microscopy (ExM), which achieves nanoscale-precise imaging of specimens at ~70 nm resolution (with ~4.5x linear expansion) by isotropic swelling of chemically processed, hydrogel-embedded tissue. ExM of C. elegans is challenged by its cuticle, which is stiff and impermeable to antibodies. Here we present a strategy, expansion of C. elegans (ExCel), to expand fixed, intact C. elegans. ExCel enables simultaneous readout of fluorescent proteins, RNA, DNA location, and anatomical structures at resolutions of ~65-75 nm (3.3-3.8x linear expansion). We also developed epitope-preserving ExCel, which enables imaging of endogenous proteins stained by antibodies, and iterative ExCel, which enables imaging of fluorescent proteins after 20x linear expansion. We demonstrate the utility of the ExCel toolbox for mapping synaptic proteins, for identifying previously unreported proteins at cell junctions, and for gene expression analysis in multiple individual neurons of the same animal

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Expansion microscopy of C. elegans : nanoscale imaging of biomolecules throughout an entire organism

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, May, 2020Cataloged from student-submitted PDF version of thesis. "March 2020." Date of graduation, May 2020.Includes bibliographical references (pages 140-146).Expansion microscopy (ExM) enables 3-D, nanoscale-precise imaging of biological specimens by isotropic swelling of hydrogel-embedded, chemically processed tissue. Such capability raises the question of whether nanoscale mapping of biomolecules could be performed in an entire organism, which would allow super-resolution-mediated in situ analyses, such as digital quantification of biomolecules and mapping of synaptic contacts, to be performed within the context of an entire nervous system. The nematode Caenorhabditis elegans could be a suitable model for such organism-wide analyses, due to its tractable physical size, deterministic cell lineage, ease of genetic control, and well-established literature. However, C. elegans is enclosed in a chemically impermeable and mechanically tough cuticle, which could hinder the deployment of ExM. In this thesis, we present a strategy, expansion of C. elegans (ExCel), to expand fixed, cuticle-enclosed intact animals of C. elegans. ExCel enables simultaneous readout of fluorescent proteins, RNAs, DNA locations, and anatomical structures at resolutions of ~65-75 nm (3.3-3.8x linear expansion). We also developed epitope-preserving ExCel, which enables imaging of endogenous proteins stained by antibodies, and iterative ExCel, which enables imaging of fluorescent proteins at a ~25-nm resolution (20x linear expansion). We demonstrate the utility of the ExCel toolbox for multiplexed imaging of multiple molecular types, for mapping synaptic proteins, for identifying previously unreported proteins at cell junctions, and for gene expression analysis in multiple individual neurons of the same animal. In addition to ExCel, we discuss two other ExM-related technologies, including tetragel, which is a highly homogeneous hydrogel network that improves the nanoscale isotropy of biological ultrastructure expanded by ExM, and stochastic arrangement of reporters in clusters (STARC), which is a strategy for recording neuronal activity at a subneurite-level resolution, in densely labeled neuronal populations. Taken together, the work presented in this thesis extends the capabilities of ExM, and lays the foundation for a comprehensive, functionally and structurally informed analysis of an entire organism, which could reveal new insights in neuroscience, organismal development, and systems biology.by Chih-Chieh (Jay) Yu.Ph. D.Ph. D. Massachusetts Institute of Technology, Department of Biological Engineerin

Saturated evanescent-wave absorption of few-layer graphene-covered side-polished single-mode fiber for all-optical switching

Using the evanescent-wave saturation effect of hydrogen-free low-temperature synthesized few-layer graphene covered on the cladding region of a side-polished single-mode fiber, a blue pump/infrared probe-based all-optical switch is demonstrated with specific wavelength-dependent probe modulation efficiency. Under the illumination of a blue laser diode at 405 nm, the few-layer graphene exhibits cross-gain modulation at different wavelengths covering the C- and L-bands. At a probe power of 0.5 mW, the L-band switching throughput power variant of 16 μW results in a probe modulation depth of 3.2%. Blue shifting the probe wavelength from 1580 to 1520 nm further enlarges the switching throughput power variant to 24 mW and enhances the probe modulation depth to 5%. Enlarging the probe power from 0.5 to 1 mW further enlarges the switching throughput power variant from 25 to 58 μW to promote its probe modulation depth of up to 5.8% at 1520 nm. In contrast, the probe modulation depth degrades from 5.1% to 1.2% as the pumping power reduces from 85 to 24 mW, which is attributed to the saturable absorption of the few-layer graphene-based evanescent-wave absorber. The modulation depth at wavelength of 1550 nm under a probe power of 1 mW increases from 1.2% to 5.1%, as more carriers can be excited when increasing the blue laser power from 24 to 85 mW, whereas it decreases from 5.1% to 3.3% by increasing the input probe power from 1 to 2 mW to show an easier saturated condition at longer wavelength

Expansion microscopy of C. elegans

© Yu et al. We recently developed expansion microscopy (ExM), which achieves nanoscale-precise imaging of specimens at ~70 nm resolution (with ~4.5x linear expansion) by isotropic swelling of chemically processed, hydrogel-embedded tissue. ExM of C. elegans is challenged by its cuticle, which is stiff and impermeable to antibodies. Here we present a strategy, expansion of C. elegans (ExCel), to expand fixed, intact C. elegans. ExCel enables simultaneous readout of fluorescent proteins, RNA, DNA location, and anatomical structures at resolutions of ~65–75 nm (3.3–3.8x linear expansion). We also developed epitope-preserving ExCel, which enables imaging of endogenous proteins stained by antibodies, and iterative ExCel, which enables imaging of fluorescent proteins after 20x linear expansion. We demonstrate the utility of the ExCel toolbox for mapping synaptic proteins, for identifying previously unreported proteins at cell junctions, and for gene expression analysis in multiple individual neurons of the same animal

Spatial Multiplexing of Fluorescent Reporters for Imaging Signaling Network Dynamics

© 2020 The Author(s) In order to analyze how a signal transduction network converts cellular inputs into cellular outputs, ideally one would measure the dynamics of many signals within the network simultaneously. We found that, by fusing a fluorescent reporter to a pair of self-assembling peptides, it could be stably clustered within cells at random points, distant enough to be resolved by a microscope but close enough to spatially sample the relevant biology. Because such clusters, which we call signaling reporter islands (SiRIs), can be modularly designed, they permit a set of fluorescent reporters to be efficiently adapted for simultaneous measurement of multiple nodes of a signal transduction network within single cells. We created SiRIs for indicators of second messengers and kinases and used them, in hippocampal neurons in culture and intact brain slices, to discover relationships between the speed of calcium signaling, and the amplitude of PKA signaling, upon receiving a cAMP-driving stimulus

Expansion sequencing: Spatially precise in situ transcriptomics in intact biological systems

Methods for highly multiplexed RNA imaging are limited in spatial resolution and thus in their ability to localize transcripts to nanoscale and subcellular compartments. We adapt expansion microscopy, which physically expands biological specimens, for long-read untargeted and targeted in situ RNA sequencing. We applied untargeted expansion sequencing (ExSeq) to the mouse brain, which yielded the readout of thousands of genes, including splice variants. Targeted ExSeq yielded nanoscale-resolution maps of RNAs throughout dendrites and spines in the neurons of the mouse hippocampus, revealing patterns across multiple cell types, layer-specific cell types across the mouse visual cortex, and the organization and position-dependent states of tumor and immune cells in a human metastatic breast cancer biopsy. Thus, ExSeq enables highly multiplexed mapping of RNAs from nanoscale to system scale.ISSN:0036-8075ISSN:1095-920