Expansion microscopy Chen, Fei; Tillberg, Paul W.; Boyden, Edward S.
Science,
01/2015, Letnik:
347, Številka:
6221
Journal Article
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In optical microscopy, fine structural details are resolved by using refraction to magnify images of a specimen. We discovered that by synthesizing a swellable polymer network within a specimen, it ...can be physically expanded, resulting in physical magnification. By covalently anchoring specific labels located within the specimen directly to the polymer network, labels spaced closer than the optical diffraction limit can be isotropically separated and optically resolved, a process we call expansion microscopy (ExM). Thus, this process can be used to perform scalable superresolution microscopy with diffraction-limited microscopes. We demonstrate ExM with apparent ∼70-nanometer lateral resolution in both cultured cells and brain tissue, performing three-color superresolution imaging of ∼107 cubic micrometers of the mouse hippocampus with a conventional confocal microscope.
Understanding genome organization requires integration of DNA sequence and three-dimensional spatial context; however, existing genome-wide methods lack either base pair sequence resolution or direct ...spatial localization. Here, we describe in situ genome sequencing (IGS), a method for simultaneously sequencing and imaging genomes within intact biological samples. We applied IGS to human fibroblasts and early mouse embryos, spatially localizing thousands of genomic loci in individual nuclei. Using these data, we characterized parent-specific changes in genome structure across embryonic stages, revealed single-cell chromatin domains in zygotes, and uncovered epigenetic memory of global chromosome positioning within individual embryos. These results demonstrate how IGS can directly connect sequence and structure across length scales from single base pairs to whole organisms.
Thoracic sarcoidosis is the most common form of sarcoidosis, encompassing a heterogeneous group of patients with a wide range of clinical features and associated outcomes. The distinction between ...isolated thoracic lymphadenopathy and pulmonary involvement matters. Morbidity is often higher, and long-term outcomes are worse for the latter. Although inflammatory infiltrates in pulmonary sarcoidosis may resolve, persistent disease activity is common and can result in lung fibrosis. Given the distinct clinical features and natural history of pulmonary sarcoidosis, its pathogenesis may differ in important ways from other sarcoidosis manifestations. This review highlights recent advances in the pathogenesis of pulmonary sarcoidosis, including the nature of the sarcoidosis antigen, the role of serum amyloid A and other host factors that contribute to alterations in innate immunity, factors that shape adaptive T-cell profiles in the lung, and how these mechanisms influence the maintenance of granulomatous inflammation in sarcoidosis. We discuss questions raised by recent findings, including the role of innate immunity in the pathogenesis, the meaning of immune cell exhaustion, and mechanisms that may contribute to lung fibrosis in sarcoidosis. We conclude with a reflection on when and how immunosuppressive therapies may be helpful for pulmonary sarcoidosis, a consideration of nonpharmacologic management strategies, and a survey of potential novel therapeutic targets for this vexing disease.
A central challenge in developing quantum computers and long-range quantum networks is the distribution of entanglement across many individually controllable qubits
. Colour centres in diamond have ...emerged as leading solid-state 'artificial atom' qubits
because they enable on-demand remote entanglement
, coherent control of over ten ancillae qubits with minute-long coherence times
and memory-enhanced quantum communication
. A critical next step is to integrate large numbers of artificial atoms with photonic architectures to enable large-scale quantum information processing systems. So far, these efforts have been stymied by qubit inhomogeneities, low device yield and complex device requirements. Here we introduce a process for the high-yield heterogeneous integration of 'quantum microchiplets'-diamond waveguide arrays containing highly coherent colour centres-on a photonic integrated circuit (PIC). We use this process to realize a 128-channel, defect-free array of germanium-vacancy and silicon-vacancy colour centres in an aluminium nitride PIC. Photoluminescence spectroscopy reveals long-term, stable and narrow average optical linewidths of 54 megahertz (146 megahertz) for germanium-vacancy (silicon-vacancy) emitters, close to the lifetime-limited linewidth of 32 megahertz (93 megahertz). We show that inhomogeneities of individual colour centre optical transitions can be compensated in situ by integrated tuning over 50 gigahertz without linewidth degradation. The ability to assemble large numbers of nearly indistinguishable and tunable artificial atoms into phase-stable PICs marks a key step towards multiplexed quantum repeaters
and general-purpose quantum processors
.
Sequencing of 16S rRNA gene tags is a popular method for profiling and comparing microbial communities. The protocols and methods used, however, vary considerably with regard to amplification ...primers, sequencing primers, sequencing technologies; as well as quality filtering and clustering. How results are affected by these choices, and whether data produced with different protocols can be meaningfully compared, is often unknown. Here we compare results obtained using three different amplification primer sets (targeting V4, V6-V8, and V7-V8) and two sequencing technologies (454 pyrosequencing and Illumina MiSeq) using DNA from a mock community containing a known number of species as well as complex environmental samples whose PCR-independent profiles were estimated using shotgun sequencing. We find that paired-end MiSeq reads produce higher quality data and enabled the use of more aggressive quality control parameters over 454, resulting in a higher retention rate of high quality reads for downstream data analysis. While primer choice considerably influences quantitative abundance estimations, sequencing platform has relatively minor effects when matched primers are used. Beta diversity metrics are surprisingly robust to both primer and sequencing platform biases.
Immunologically, sarcoidosis has been classically defined by the presence of a Th1-dominant environment localised to sites of disease, characterised by overexpression of interferon (IFN)γ and ...Th1-promoting interleukin (IL)12 and IL18 1. Accordingly, the downstream effects of enhanced Th1 are also observed in sarcoidosis, such as the expression of Th1-associated chemokines (MIG/CXCL9, IP10/CXCL10, ITAC/CXCL11, MCP1/CCL2, MIP1α/CCL3, MIP1β/CCL4, RANTES/CCL5) and their respective receptors (CXCR3, CCR5). Conversely, the presence of augmented Th2 has never been firmly documented in the sarcoidosis lung.
Maize is an important crop with a high level of genome diversity and heterosis. The genome sequence of a typical female line, B73, was previously released. Here, we report a de novo genome assembly ...of a corresponding male representative line, Mo17. More than 96.4% of the 2,183 Mb assembled genome can be accounted for by 362 scaffolds in ten pseudochromosomes with 38,620 annotated protein-coding genes. Comparative analysis revealed large gene-order and gene structural variations: approximately 10% of the annotated genes were mutually nonsyntenic, and more than 20% of the predicted genes had either large-effect mutations or large structural variations, which might cause considerable protein divergence between the two inbred lines. Our study provides a high-quality reference-genome sequence of an important maize germplasm, and the intraspecific gene order and gene structural variations identified should have implications for heterosis and genome evolution.
Lithographic nanofabrication is often limited to successive fabrication of two-dimensional (2D) layers. We present a strategy for the direct assembly of 3D nanomaterials consisting of metals, ...semiconductors, and biomolecules arranged in virtually any 3D geometry. We used hydrogels as scaffolds for volumetric deposition of materials at defined points in space. We then optically patterned these scaffolds in three dimensions, attached one or more functional materials, and then shrank and dehydrated them in a controlled way to achieve nanoscale feature sizes in a solid substrate. We demonstrate that our process, Implosion Fabrication (ImpFab), can directly write highly conductive, 3D silver nanostructures within an acrylic scaffold via volumetric silver deposition. Using ImpFab, we achieve resolutions in the tens of nanometers and complex, non-self-supporting 3D geometries of interest for optical metamaterials.
Expansion microscopy (ExM) enables imaging of preserved specimens with nanoscale precision on diffraction-limited instead of specialized super-resolution microscopes. ExM works by physically ...separating fluorescent probes after anchoring them to a swellable gel. The first ExM method did not result in the retention of native proteins in the gel and relied on custom-made reagents that are not widely available. Here we describe protein retention ExM (proExM), a variant of ExM in which proteins are anchored to the swellable gel, allowing the use of conventional fluorescently labeled antibodies and streptavidin, and fluorescent proteins. We validated and demonstrated the utility of proExM for multicolor super-resolution (∼70 nm) imaging of cells and mammalian tissues on conventional microscopes.
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