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3920 Publications
Showing 3301-3310 of 3920 resultsSignaling through the TNF-family receptor Fas/CD95 can trigger apoptosis or non-apoptotic cellular responses and is essential for protection from autoimmunity. Receptor clustering has been observed following interaction with Fas ligand (FasL), but the stoichiometry of Fas, particularly when triggered by membrane-bound FasL, the only form of FasL competent at inducing programmed cell death, is not known. Here we used super-resolution microscopy to study the behavior of single molecules of Fas/CD95 on the plasma membrane after interaction of Fas with FasL on planar lipid bilayers. We observed rapid formation of Fas protein superclusters containing more than 20 receptors after interactions with membrane-bound FasL. Fluorescence correlation imaging demonstrated recruitment of FADD dependent on an intact Fas death domain, with lipid raft association playing a secondary role. Flow-cytometric FRET analysis confirmed these results, and also showed that some Fas clustering can occur in the absence of FADD and caspase-8. Point mutations in the Fas death domain associated with autoimmune lymphoproliferative syndrome (ALPS) completely disrupted Fas reorganization and FADD recruitment, confirming structure-based predictions of the critical role that these residues play in Fas-Fas and Fas-FADD interactions. Finally, we showed that induction of apoptosis correlated with the ability to form superclusters and recruit FADD.
Microscopic resolution far beyond the diffraction limit is possible by localizing single molecules individually. This approach has now been demonstrated on living cells.
Arrays of actin filaments (F-actin) near the apical surface of epithelial cells (medioapical arrays) contribute to apical constriction and morphogenesis throughout phylogeny. Here, super-resolution approaches (grazing incidence structured illumination, GI-SIM and lattice light sheet, LLSM) microscopy resolve individual, fluorescently labeled F-actin and bipolar myosin filaments that drive amnioserosa cell shape changes during dorsal closure in . In expanded cells, F-actin and myosin form loose, apically domed meshworks at the plasma membrane. The arrays condense as cells contract, drawing the domes into the plane of the junctional belts. As condensation continues, individual filaments are no longer uniformly apparent. As cells expand, arrays of actomyosin are again resolved - some F-actin turnover likely occurs, but a large fraction of existing filaments rearrange. In morphologically isotropic cells, actin filaments are randomly oriented and during contraction, are drawn together but remain essentially randomly oriented. In anisotropic cells, largely parallel actin filaments are drawn closer to one another. Our images offer unparalleled resolution of F-actin in embryonic tissue show that medioapical arrays are tightly apposed to the plasma membrane, are continuous with meshworks of lamellar F-actin and thereby constitute modified cell cortex. In concert with other tagged array components, super-resolution imaging of live specimens will offer new understanding of cortical architecture and function. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].
Quantitative retinal imaging is essential for advanced study and clinical management of eye diseases. However, spatial resolution of retinal imaging has been limited due to available numerical aperture and optical aberration of the ocular optics. Structured illumination microscopy has been established to break the diffraction-limit resolution in conventional light microscopy. However, practical implementation of structured illumination microscopy for ophthalmoscopy of the retina is challenging due to inevitable eye movements that can produce phase artifacts. Recently, we have demonstrated the feasibility of using virtually structured detection as one alternative to structured illumination microscopy for super-resolution imaging. By providing the flexibility of digital compensation of eye movements, the virtually structured detection provides a feasible, phase-artifact-free strategy to achieve super-resolution ophthalmoscopy. In this article, we summarize the technical rationale of virtually structured detection, and its implementations for super-resolution imaging of freshly isolated retinas, intact animals, and awake human subjects.
Traditional photon localization microscopy analyses only the spatial distributions of photons emitted by individual molecules to reconstruct super-resolution optical images. Unfortunately, however, the highly valuable spectroscopic information from these photons have been overlooked. Here we report a spectroscopic photon localization microscopy that is capable of capturing the inherent spectroscopic signatures of photons from individual stochastic radiation events. Spectroscopic photon localization microscopy achieved higher spatial resolution than traditional photon localization microscopy through spectral discrimination to identify the photons emitted from individual molecules. As a result, we resolved two fluorescent molecules, which were 15 nm apart, with the corresponding spatial resolution of 10 nm-a four-fold improvement over photon localization microscopy. Using spectroscopic photon localization microscopy, we further demonstrated simultaneous multi-colour super-resolution imaging of microtubules and mitochondria in COS-7 cells and showed that background autofluorescence can be identified through its distinct emission spectra.
A central problem in neuroscience is reconstructing neuronal circuits on the synapse level. Due to a wide range of scales in brain architecture such reconstruction requires imaging that is both high-resolution and high-throughput. Existing electron microscopy (EM) techniques possess required resolution in the lateral plane and either high-throughput or high depth resolution but not both. Here, we exploit recent advances in unsupervised learning and signal processing to obtain high depth-resolution EM images computationally without sacrificing throughput. First, we show that the brain tissue can be represented as a sparse linear combination of localized basis functions that are learned using high-resolution datasets. We then develop compressive sensing-inspired techniques that can reconstruct the brain tissue from very few (typically 5) tomographic views of each section. This enables tracing of neuronal processes and, hence, high throughput reconstruction of neural circuits on the level of individual synapses.
Structured-illumination microscopy can double the resolution of the widefield fluorescence microscope but has previously been too slow for dynamic live imaging. Here we demonstrate a high-speed structured-illumination microscope that is capable of 100-nm resolution at frame rates up to 11 Hz for several hundred time points. We demonstrate the microscope by video imaging of tubulin and kinesin dynamics in living Drosophila melanogaster S2 cells in the total internal reflection mode.
The three-dimensional (3D) genome structure plays a fundamental role in gene regulation and cellular functions. Recent studies in 3D genomics inferred the very basic functional chromatin folding structures known as chromatin loops, the long-range chromatin interactions that are mediated by protein factors and dynamically extruded by cohesin. We combined the use of FISH staining of a very short (33 kb) chromatin fragment, interferometric photoactivated localization microscopy (iPALM), and traveling salesman problem-based heuristic loop reconstruction algorithm from an image of the one of the strongest CTCF-mediated chromatin loops in human lymphoblastoid cells. In total, we have generated thirteen good quality images of the target chromatin region with 2-22 nm oligo probe localization precision. We visualized the shape of the single chromatin loops with unprecedented genomic resolution which allowed us to study the structural heterogeneity of chromatin looping. We were able to compare the physical distance maps from all reconstructed image-driven computational models with contact frequencies observed by ChIA-PET and Hi-C genomic-driven methods to examine the concordance between single cell imaging and population based genomic data.
Biological tissues are rarely transparent, presenting major challenges for deep tissue optical microscopy. The achievable imaging depth is fundamentally limited by wavefront distortions caused by aberration and random scattering. Here, we report an iterative wavefront compensation technique that takes advantage of the nonlinearity of multiphoton signals to determine and compensate for these distortions and to focus light inside deep tissues. Different from conventional adaptive optics methods, this technique can rapidly measure highly complicated wavefront distortions encountered in deep tissue imaging and provide compensations for not only aberration but random scattering. The technique is tested with a variety of highly heterogeneous biological samples including mouse brain tissue, skull, and lymph nodes. We show that high quality three-dimensional imaging can be realized at depths beyond the reach of conventional multiphoton microscopy and adaptive optics methods, albeit over restricted distances for a given correction. Moreover, the required laser excitation power can be greatly reduced in deep tissues, deviating from the power requirement of ballistic light excitation and thus significantly reducing photo damage to the biological tissue.
While it is clear that key transcriptional programmes are important for maintaining pluripotency, the requirement for cell adhesion to the extracellular matrix remains poorly defined. Human pluripotent stem cells (hPSCs) form colonies encircled by an actin ring and large stable cornerstone focal adhesions (FA). Using superresolution two-colour interferometric photo-activated localisation microscopy, we examine the three-dimensional architecture of cornerstone adhesions and report vertical lamination of FA proteins with three main structural features distinct from previously studied focal adhesions: 1) integrin β5 and talin are present at high density, at the edges of cornerstone FA, adjacent to a vertical kank-rich protein wall, 2) vinculin localises higher than previously reported, displaying a head-above-tail orientation, and 3) surprisingly, actin and α-actinin are present in two discrete z-layers. Finally, we report that depletion of kanks diminishes FA patterning, and actin organisation within the colony, indicating a role for kanks in hPSC colony architecture.