分类: 生物学 >> 生物物理学 提交时间: 2016-05-12
摘要: We demonstrate the use of cryogenic super-resolution correlative light and electron microscopy (csCLEM) to precisely determine the spatial relationship between proteins and their native cellular structures. Several fluorescent proteins (FPs) were found to be photoswitchable and emitted far more photons under our cryogenic imaging condition, resulting in higher localization precision which is comparable to ambient super-resolution imaging. Vitrified specimens were prepared by high pressure freezing and cryo-sectioning to maintain a near-native state with better fluorescence preservation. A 2-3-fold improvement of resolution over the recent reports was achieved due to the photon budget performance of screening out Dronpa and optimized imaging conditions, even with thin sections which is at a disadvantage when calculate the structure resolution from label density. We extended csCLEM to mammalian cells by introducing cryo-sectioning and observed good correlation of a mitochondrial protein with the mitochondrial outer membrane at nanometer resolution in three dimensions.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-12
摘要: Super-resolution microscopy techniques have overcome the limit of optical diffraction. Recently, the Bayesian analysis of Bleaching and Blinking data (3B) method has emerged as an important tool to obtain super-resolution fluorescence images. 3B uses the change in information caused by adding or removing fluorophores in the cell to fit the data. When adding a new fluorophore, 3B selects a random initial position, optimizes this position and then determines its reliability. However, the fluorophores are not evenly distributed in the entire image region, and the fluorescence intensity at a given position positively correlates with the probability of observing a fluorophore at this position. In this paper, we present a Bayesian analysis of Bleaching and Blinking microscopy method based on fluorescence intensity distribution (FID3B). We utilize the intensity distribution to select more reliable positions as the initial positions of fluorophores. This approach can improve the reconstruction results and significantly reduce the computational time. We validate the performance of our method using both simulated data and experimental data from cellular structures. The results confirm the effectiveness of our method.
分类: 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间: 2016-05-05
摘要: Autofocusing is a fundamental step towards automated microscopic screening of Caenorhabditis elegans. Determining the optimal focus in an optical microscope is based on a clarity-evaluation function that is applied to images acquired from different focuses of the same field. The maximum value of the function is considered as the point of optimal focus. In this paper, 16 autofocus algorithms which were collected from well-known algorithms as well as the most recently proposed focusing algorithms have been evaluated. Through these evaluations, an optimal algorithm was found for C. elegants lipid droplets to set up an automatic screening system. Many features were assessed in this paper, for instance accuracy, computational time, addition of noise, and focusing curve. Our results have shown that most of the algorithms show an overall high performance for this type of image, and absolute Tenengrad algorithm will be our first choice for its best performance considering accuracy.
分类: 生物学 >> 生物物理学 提交时间: 2016-05-12
摘要: Reversibly switchable fluorescent proteins (RSFPs) can be effectively used for super-resolution optical fluctuation imaging (SOFI) based on the switching and fluctuation of single molecules. Several properties of RSFPs strongly influence the quality of SOFI images. These properties include (i) the averaged fluorescence intensity in the fluctuation state, (ii) the on/off contrast ratio, (iii) the photostability, and (iv) the oligomerization tendency. The first three properties determine the fluctuation range of the imaged pixels and the SOFI signal, which are of essential importance to the spatial resolution, and the last may lead to artificial aggregation of target proteins. The RSFPs that are currently used for SOFI are low in averaged fluorescence intensity in the fluctuation state, photostability, and on/off contrast ratio, thereby limiting the range of application of SOFI in biological super-resolution imaging. In this study, we developed a novel monomeric green RSFP termed Skylan-S, which features very high photostability, contrast ratio, and averaged fluorescence intensity in the fluctuation state. Taking advantage of the excellent optical properties of Skylan-S, a 4-fold improvement in the fluctuation range of the imaged pixels and higher SOFI resolution can be obtained compared with Dronpa. Furthermore, super-resolution imaging of the actin or tubulin structures and clathrin-coated pits (CCPs) in living U2OS cells labeled with Skylan-S was demonstrated using the SOFI technique. Overall, Skylan-S developed with outstanding photochemical properties is promising for long-time SOH imaging with high spatial-temporal resolution.
分类: 光学 >> 显微镜 提交时间: 2023-11-15
摘要: Afull-field transmissionhardX-ray microscope (TXM) with 30nm resolution was designed and its prototype was constructed. The TXM relies on a compact, high stiffness, low heat dissipation and low vibration design philosophy and utilizes Fresnel Zone plate (FZP) as imaging optics. The design of the TXMwas introduced in detail, including the optical layout, the parameters of the FZP, the mechanical design of the TXM instrument. Preliminary imaging result with 52nm spatial resolution was achieved.
分类: 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间: 2016-05-11
摘要: Background: Phosphatidylinositides in the plasma membrane (PM) are pivotal for cellular functions. Results: Superresolution imaging reveals homogeneous distribution of PI(4,5)P-2, PI4P, and PI(3,4,5)P-3 in the major area of the PM. Conclusion: Phosphatidylinositides detected by PH domains are uniformly distributed in the major regions of the PM, with limited concentration gradients. Significance: This result may imply a new working model of phosphatidylinositides at nanometer scale. Both phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-2) are independent plasma membrane (PM) determinant lipids that are essential for multiple cellular functions. However, their nanoscale spatial organization in the PM remains elusive. Using single-molecule superresolution microscopy and new photoactivatable fluorescence probes on the basis of pleckstrin homology domains that specifically recognize phosphatidylinositides in insulin-secreting INS-1 cells, we report that the PI(4,5)P-2 probes exhibited a remarkably uniform distribution in the major regions of the PM, with some sparse PI(4,5)P-2-enriched membrane patches/domains of diverse sizes (383 +/- 14 nm on average). Quantitative analysis revealed a modest concentration gradient that was much less steep than previously thought, and no densely packed PI(4,5)P-2 nanodomains were observed. Live-cell superresolution imaging further demonstrated the dynamic structural changes of those domains in the flat PM and membrane protrusions. PI4P and phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P-3) showed similar spatial distributions as PI(4,5)P-2. These data reveal the nanoscale landscape of key inositol phospholipids in the native PM and imply a framework for local cellular signaling and lipid-protein interactions at a nanometer scale.
分类: 核科学技术 >> 辐射物理与技术 提交时间: 2023-11-22
摘要: Full-field transmission X-ray microscopy (TXM) is a powerful nondestructive three-dimensional (3D) imaging method with a nanoscale spatial resolution that has been used in most synchrotron facilities worldwide. An in-house-designed TXM system was constructed at the BL18B 3D Nanoimaging beamline at the Shanghai Synchrotron Radiation Facility. The beamline operates from 5 to 14 keV and enables 20 nm spatial resolution imaging. The characterization details of the beamline are described in this paper. The performances in terms of spatial resolution, nano-CT, and nano-spectral imaging of the TXM beamline are also presented in this article.
分类: 物理学 >> 普通物理:统计和量子力学,量子信息等 提交时间: 2017-05-02
摘要: Titania-hydroxyapatite (HA) nanocomposites were fabricated by wet chemical synthesis approach. HA exhibited crystallographic orientation of nucleation on nano titania particle, forming the composite particles with titania being partially enwrapped with HA. Microstructural characterization by high res- olution transmission electron microscopy revealed coherent interfacial bond of (110) and (222) planes of HA crystal with (101) plane of anatase. The HA layer promoted significantly recruitment of Escherichia coli bacteria onto the titania-based particles for subsequent photocatalytic killing. Less extent of enwrapping of HA on titania particle, as accomplished by increasing the aging time of HA suspension, gave rise to better capability of photocatalytic degradation of methylene blue and sterilization of the bacteria. The novel HA-enwrapped titania powder shows great potential for environmental applications.
分类: 地球科学 >> 地质学 提交时间: 2017-08-29 合作期刊: 《古脊椎动物学报》
摘要: Synchrotron Radiation X-ray Tomographic Microscopy (SRXTM) is a powerful non-destructive method in paleontology, providing ultra-high-resolution 3D insights into the internal structure of fossils. Employing SRXTM, the skull specimens of Shuyu zhejiangensis, a 428 million-year-old galeaspid from the Silurian of Changxing, Zhejiang Province, are investigated. The subsequent analyses indicate that the endoskeletal skull of S. zhejiangensis is composed wholly of cartilage without convincing evidence for the presence of perichondral bone. The cranial anatomy of S. zhejiangensis are unusually preserved in three dimensions largely due to the non-random decay of the cartilaginous braincase and its connecting ‘soft’ tissues. Using Amira or Avizo software, seven virtual 3D endocasts of the skull of S. zhejiangensis were created revealing the gross internal cranial anatomy of galeaspids in great detail for the first time. The preliminary results indicate that during evolution the galeaspid head experienced a fundamental reorganization resulting in the development of jaws. 同步辐射X射线断层显微成像已经成为古生物研究中一种新的重要手段,能够在不损坏化石的前提下,提供化石内部超高分辨率的三维成像。利用瑞士光源最先进的同步辐射X射线断层显微成像技术,研究分析了七个采自浙江长兴志留系(约4.28亿年前)的早期盔甲鱼类浙江曙鱼三维立体保存脑颅化石。同步辐射X射线显微成像结果显示:盔甲鱼脑颅完全由软骨组成,并没有软骨外成骨存在的证据;盔甲鱼脑颅解剖结构能够被精细的保存下来,很大程度上归功于脑颅软骨与周围软组织在埋藏过程中的异时分解。利用AMIRA或AVIZO等计算机三维虚拟复原软件,三维虚拟复原了七个曙鱼脑颅模型,首次揭示出盔甲鱼脑颅内部详细的解剖结构。初步研究结果显示盔甲鱼的脑颅已经发生了显著的重组,具备了颌发育所必需的先决条件。
分类: 核科学技术 >> 粒子加速器 提交时间: 2023-06-18 合作期刊: 《Nuclear Science and Techniques》
摘要: With the spatial coherence of X-rays and high flux and brightness of the 3rd generation synchrotron radiation facility, X-ray phase contrast microscopy (XPCM) at Shanghai Synchrotron Radiation Facility (SSRF) can provide high resolution dynamic imaging of low electron density materials in principle. In this paper, we investigated the cavitation and water-refilling processes in rice and bamboo leaves utilizing XPCM at SSRF. The occurrence of xylem cavitation was recorded in vivo. The study also revealed that under different dehydration conditions, cavitation occurs in different degrees, and therefore, the refilling process is different. The results demonstrate that SSRF can provide high enough fluxes to study dynamic processes in plants in real-time, and XPCM is expected to be a promising method to reveal the mechanisms of cavitation and its repair in plants nondestructively.
分类: 物理学 >> 普通物理:统计和量子力学,量子信息等 提交时间: 2017-05-02
摘要: Polypropylene and calcium carbonate were prepared by melt extrusion in a twin screw extruder or cast- ing in a kneader extruder. The dispersion of CaCO3 particles in PP was analysed through the observation of impact-fracture surface, polished surface, cross-section surface and film using scanning electron microscopy (SEM), focused ion beam (FIB) and optical transmission microscopy (OTM). Only basic infor- mation on particle dispersion was obtained from the investigation of fracture surfaces by SEM and the FIB cross-sections by virtue of the ambiguous phase contrast and the limited area observed. However, the SEM images from polished-surfaces and the film images observed in OTM show the different particle dispersion in different samples. The derived Particle Size Distribution (PSD) curves from OTM images explain the difference of impact strength, demonstrating the relationship between particle dispersion and mechanical properties.
分类: 物理学 >> 普通物理:统计和量子力学,量子信息等 提交时间: 2017-05-02
摘要: Silver nanoparticles with a mean diameter of approximately 30 nm were synthesized by reduction of silver nitrate with triethanolamine in the presence of polyacrylic acid. Silver nanoparticle-based ink was prepared by dispersing silver nanoparticles into a mixture of water and ethylene glycol. The mechanism for the dispersion and aggregation of silver nanoparticles in ink is discussed. The strong electrostatic repulsions of the carboxylate anions of the adsorbed polyacrylic acid molecules disturbed the aggrega- tion of metal particles in solutions with a high pH value (pH > 5). An inkjet printer was used to deposit this silver nanoparticle-based ink to form silver patterns on photo paper. The actual printing qualities of the silver tracks were then analyzed by variation of printing passes, sintering temperature and time. The results showed that sintering temperature and time are associated strongly with the conductivity of the inkjet-printed conductive patterns. The conductivity of printed patterns sintered at 150 C increased to 2.1 107 S m 1, which was approximately one third that of bulk silver. In addition, silver tracks on paper substrate also showed better electrical performance after folding. This study demonstrated that the resulting ink-jet printed patterns can be used as conductive tracks in flexible electronic devices.
分类: 生物学 >> 生物物理学 提交时间: 2016-05-11
摘要: Background: The recent developments of far-field optical microscopy (single molecule imaging techniques) have overcome the diffraction barrier of light and improve image resolution by a factor of ten compared with conventional light microscopy. These techniques utilize the stochastic switching of probe molecules to overcome the diffraction limit and determine the precise localizations of molecules, which often requires a long image acquisition time. However, long acquisition times increase the risk of sample drift. In the case of high resolution microscopy, sample drift would decrease the image resolution. Results: In this paper, we propose a novel metric based on the distance between molecules to solve the drift correction. The proposed metric directly uses the position information of molecules to estimate the frame drift. We also designed an algorithm to implement the metric for the general application of drift correction. There are two advantages of our method: First, because our method does not require space binning of positions of molecules but directly operates on the positions, it is more natural for single molecule imaging techniques. Second, our method can estimate drift with a small number of positions in each temporal bin, which may extend its potential application. Conclusions: The effectiveness of our method has been demonstrated by both simulated data and experiments on single molecular images.
分类: 生物学 >> 生物物理学 提交时间: 2016-05-11
摘要: Identification and detection of dendritic spines in neuron images are of high interest in diagnosis and treatment of neurological and psychiatric disorders (e.g., Alzheimer's disease, Parkinson's diseases, and autism). In this paper, we have proposed a novel automatic approach using wavelet-based conditional symmetric analysis and regularized morphological shared-weight neural networks (RMSNN) for dendritic spine identification involving the following steps: backbone extraction, localization of dendritic spines, and classification. First, a new algorithm based on wavelet transform and conditional symmetric analysis has been developed to extract backbone and locate the dendrite boundary. Then, the RMSNN has been proposed to classify the spines into three predefined categories (mushroom, thin, and stubby). We have compared our proposed approach against the existing methods. The experimental result demonstrates that the proposed approach can accurately locate the dendrite and accurately classify the spines into three categories with the accuracy of 99.1% for "mushroom" spines, 97.6% for "stubby" spines, and 98.6% for "thin" spines.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-05
摘要: Single particle analysis, which can be regarded as an average of signals from thousands or even millions of particle projections, is an efficient method to study the three-dimensional structures of biological macromolecules. An intrinsic assumption in single particle analysis is that all the analyzed particles must have identical composition and conformation. Thus specimen heterogeneity in either composition or conformation has raised great challenges for high-resolution analysis. For particles with multiple conformations, inaccurate alignments and orientation parameters will yield an averaged map with diminished resolution and smeared density. Besides extensive classification approaches, here based on the assumption that the macromolecular complex is made up of multiple rigid modules whose relative orientations and positions are in slight fluctuation around equilibriums, we propose a new method called as local optimization refinement to address this conformational heterogeneity for an improved resolution. The key idea is to optimize the orientation and shift parameters of each rigid module and then reconstruct their three-dimensional structures individually. Using simulated data of 80S/70S ribosomes with relative fluctuations between the large (60S/50S) and the small (40S/30S) subunits, we tested this algorithm and found that the resolutions of both subunits are significantly improved. Our method provides a proof-of-principle solution for high-resolution single particle analysis of macromolecular complexes with dynamic conformations.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-11
摘要: Many receptor-mediated endocytic processes are mediated by constitutive budding of clathrin-coated pits (CCPs) at spatially randomized sites before slowly pinching off from the plasma membrane (60100 s). In contrast, clathrin-mediated endocytosis (CME) coupled with regulated exocytosis in excitable cells occurs at peri-exocytic sites shortly after vesicle fusion (similar to 10 s). The molecular mechanism underlying this spatiotemporal coupling remains elusive. We show that coupled endocytosis makes use of preformed CCPs, which hop to nascent fusion sites nearby following vesicle exocytosis. A dynamic cortical microtubular network, anchored at the cell surface by the cytoplasmic linker-associated protein on microtubules and the LL5 beta/ELKS complex on the plasma membrane, provides the track for CCP hopping. Local diacylglycerol gradients generated upon exocytosis guide the direction of hopping. Overall, the CCP-cytoskeleton-lipid interaction demonstrated here mediates exocytosis-coupled fast recycling of both plasma membrane and vesicular proteins, and it is required for the sustained exocytosis during repetitive stimulations.
分类: 生物学 >> 生物物理学 提交时间: 2016-05-11
摘要: Maize chlorotic mottle virus (MCMV) is the only member of the Machlomovirus genus in the family Tombusviridae. Here, we obtained the Cryo-EM structure of MCMV by single particle analysis with most local resolution at approximately 4 angstrom. The C alpha backbone was built based on residues with bulky side chains. The resolved C-terminus of the capsid protein subunit and obvious openings at the 2-fold axis demonstrated the compactness of the asymmetric unit, which indicates an important role in the stability of MCMV. The Asp116 residue from each subunit around the 5-fold and 3-fold axes contributed to the negative charges in the centers of the pentamers and hexamers, which might serve as a solid barrier against the leakage of genomic RNA. Finally, the loops most exposed on the surface were analyzed and are proposed to be potential functional sites related to MCMV transmission. (C) 2015 Elsevier Inc. All rights reserved.
分类: 生物学 >> 生物物理学 >> 免疫学 提交时间: 2016-05-11
摘要: Hemocyanins present in invertebrate hemolymph are multifunctional proteins, responsible for oxygen transport and contributing to innate immunity through phenoloxidase-like activity. In arthropods, hemocyanin has been identified as a source of broad-spectrum antimicrobial peptides during infection. Conversely, no hemocyanin-derived antimicrobial peptides have been reported for molluscs. The present study describes a putative antimicrobial region, termed haliotisin, located within the linking sequence between the alpha-helical domain and beta-sheet domain of abalone (Haliotis tuberculata) hemocyanin functional unit E. A series of synthetic peptides based on overlapping fragments of the haliotisin region were tested for their bactericidal potential. Incubating Gram-positive and Gram-negative bacteria in the presence of certain haliotisin peptides, notably peptides 3-4-5 (DTFDYKKFGYRYDSLELEGRS ISRIDELIQQRQEKDRTFAGFLLKGFGTSAS) led to reductions in microbial growth. Furthermore, transmission electron micrographs of haliotisin-treated bacteria revealed damages to the microbial cell wall. Data discussed here provides the first evidence to suggest that molluscan hemocyanin may act as a source of anti-infective peptides. (C) 2014 Elsevier Ltd. All rights reserved.
提交时间: 2017-05-02
摘要: For the high-energy battery using Li metal as a negative electrode, the electrolyte is one of the most critical factors that significantly affects the cell performance. Herein, new 75Li[2]S center dot [25-x] P2S5 center dot xP[2]O[5] [mol%] solid state el
分类: 生物学 >> 生物物理学 提交时间: 2016-05-12
摘要: Total internal reflection fluorescence microscope has often been used to study the molecular mechanisms underlying vesicle exocytosis. However, the spatial occurrence of the fusion events within a single cell is not frequently explored due to the lack of sensitive and accurate computer-assisted programs to analyze large image data sets. Here, we have developed an image analysis platform for the nonbiased identification of different types of vesicle fusion events with high accuracy in different cell types. By performing spatiotemporal analysis of stimulus-evoked exocytosis in insulin-secreting INS-1 cells, we statistically prove that individual vesicle fusion events are clustered at hotspots. This spatial pattern disappears upon the disruption of either the actin or the microtubule network; this disruption also severely inhibits evoked exocytosis. By demonstrating that newcomer vesicles are delivered from the cell interior to the surface membrane for exocytosis, we highlight a previously unappreciated mechanism in which the cytoskeleton-dependent transportation of secretory vesicles organizes exocytosis hotspots in endocrine cells.