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1.
A key obstacle in uncovering the orchestration between molecular and cellular events is the vastly different length scales on which they occur. We describe here a methodology for ultrastructurally mapping regions of cells and tissue as large as 1 mm(2) at nanometer resolution. Our approach employs standard transmission electron microscopy, rapid automated data collection, and stitching to create large virtual slides. It greatly facilitates correlative light-electron microscopy studies to relate structure and function and provides a genuine representation of ultrastructural events. The method is scalable as illustrated by slides up to 281 gigapixels in size. Here, we applied virtual nanoscopy in a correlative light-electron microscopy study to address the role of the endothelial glycocalyx in protein leakage over the glomerular filtration barrier, in an immunogold labeling study of internalization of oncolytic reovirus in human dendritic cells, in a cryo-electron microscopy study of intact vitrified mouse embryonic cells, and in an ultrastructural mapping of a complete zebrafish embryo slice.  相似文献   

2.
A novel method to flexibly fit atomic structures into electron microscopy (EM) maps using molecular dynamics simulations is presented. The simulations incorporate the EM data as an external potential added to the molecular dynamics force field, allowing all internal features present in the EM map to be used in the fitting process, while the model remains fully flexible and stereochemically correct. The molecular dynamics flexible fitting (MDFF) method is validated for available crystal structures of protein and RNA in different conformations; measures to assess and monitor the fitting process are introduced. The MDFF method is then used to obtain high-resolution structures of the E. coli ribosome in different functional states imaged by cryo-EM.  相似文献   

3.
Three-dimensional image reconstructions of large-scale protein aggregates are routinely determined by electron microscopy (EM). We combine low-resolution EM data with high-resolution structures of proteins determined by x-ray crystallography. A set of visualization and analysis procedures, termed the Situs package, has been developed to provide an efficient and robust method for the localization of protein subunits in low-resolution data. Topology-representing neural networks are employed to vector-quantize and to correlate features within the structural data sets. Microtubules decorated with kinesin-related ncd motors are used as model aggregates to demonstrate the utility of this package of routines. The precision of the docking has allowed for the extraction of unique conformations of the macromolecules and is limited only by the reliability of the underlying structural data.  相似文献   

4.
For a variety of problems in structural biology, low-resolution maps generated by electron microscopy imaging are often interpreted with the help of various flexible-fitting computational algorithms. In this work, we systematically analyze the quality of final models of various proteins obtained via molecular dynamics flexible fitting (MDFF) by varying the map-resolution, strength of structural restraints, and the steering forces. We find that MDFF can be extended to understand conformational changes in lower-resolution maps if larger structural restraints and lower steering forces are used to prevent overfitting. We further show that the capabilities of MDFF can be extended by combining it with an enhanced conformational sampling method, temperature-accelerated molecular dynamics (TAMD). Specifically, either TAMD can be used to generate better starting configurations for MDFF fitting or TAMD-assisted MDFF (TAMDFF) can be performed to accelerate conformational search in atomistic simulations.  相似文献   

5.
6.
DNA electron microscopy   总被引:8,自引:0,他引:8  
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7.
The freeze-fracture technique consists of physically breaking apart (fracturing) a frozen biological sample; structural detail exposed by the fracture plane is then visualized by vacuum-deposition of platinum-carbon to make a replica for examination in the transmission electron microscope. The four key steps in making a freeze-fracture replica are (i) rapid freezing, (ii) fracturing, (iii) replication and (iv) replica cleaning. In routine protocols, a pretreatment step is carried out before freezing, typically comprising fixation in glutaraldehyde followed by cryoprotection with glycerol. An optional etching step, involving vacuum sublimation of ice, may be carried out after fracturing. Freeze fracture is unique among electron microscopic techniques in providing planar views of the internal organization of membranes. Deep etching of ultrarapidly frozen samples permits visualization of the surface structure of cells and their components. Images provided by freeze fracture and related techniques have profoundly shaped our understanding of the functional morphology of the cell.  相似文献   

8.
High-voltage electron microscopy   总被引:1,自引:0,他引:1  
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9.
10.
Bridging fluorescence microscopy and electron microscopy   总被引:1,自引:1,他引:0  
Development of new fluorescent probes and fluorescence microscopes has led to new ways to study cell biology. With the emergence of specialized microscopy units at most universities and research centers, the use of these techniques is well within reach for a broad research community. A major breakthrough in fluorescence microscopy in biology is the ability to follow specific targets on or in living cells, revealing dynamic localization and/or function of target molecules. One of the inherent limitations of fluorescence microscopy is the resolution. Several efforts are undertaken to overcome this limit. The traditional and most well-known way to achieve higher resolution imaging is by electron microscopy. Moreover, electron microscopy reveals organelles, membranes, macromolecules, and thus aids in the understanding of cellular complexity and localization of molecules of interest in relation to other structures. With the new probe development, a solid bridge between fluorescence microscopy and electron microscopy is being built, even leading to correlative imaging. This connection provides several benefits, both scientifically as well as practically. Here, I summarize recent developments in bridging microscopy.  相似文献   

11.
12.
Several approaches have been introduced to interpret, in terms of high-resolution structure, low-resolution structural data as obtained from cryo-EM. As conformational changes are often observed in biological molecules, these techniques need to take into account the flexibility of proteins. Flexibility has been described in terms of movement between rigid domains and between rigid secondary structure elements, which present some limitations for studying dynamical properties. Normal mode analysis has also been used, but is limited to medium resolution data. All-atom molecular dynamics fitting techniques are more appropriate to fit structures into higher-resolution data as full protein flexibility is considered, but are cumbersome in terms of computational time. Here, we introduce a coarse-grained approach; a Go-model was used to represent biological molecules, combined with biased molecular dynamics to reproduce accurately conformational transitions. Illustrative examples on simulated data are shown. Accurate fittings can be obtained for resolution ranging from 5 to 20 Å. The approach was also tested on experimental data of Elongation Factor G and Escherichia coli RNA polymerase, where its validity is compared to previous models obtained from different techniques. This comparison demonstrates that quantitative flexible techniques, as opposed to manual docking, need to be considered to interpret low-resolution data.  相似文献   

13.
Electron microscopy (EM) in combination with image analysis is a powerful technique to study protein structures at low, medium, and high resolution. Since electron micrographs of biological objects are very noisy, improvement of the signal-to-noise ratio by image processing is an integral part of EM, and this is performed by averaging large numbers of individual projections. Averaging procedures can be divided into crystallographic and non-crystallographic methods. The crystallographic averaging method, based on two-dimensional (2D) crystals of (membrane) proteins, yielded in solving atomic protein structures in the last century. More recently, single particle analysis could be extended to solve atomic structures as well. It is a suitable method for large proteins, viruses, and proteins that are difficult to crystallize. Because it is also a fast method to reveal the low-to-medium resolution structures, the impact of its application is growing rapidly. Technical aspects, results, and possibilities are presented.  相似文献   

14.
Researchers have used transmission electron microscopy (TEM) to make contributions to cell biology for well over 50 years, and TEM continues to be an important technology in our field. We briefly present for the neophyte the components of a TEM-based study, beginning with sample preparation through imaging of the samples. We point out the limitations of TEM and issues to be considered during experimental design. Advanced electron microscopy techniques are listed as well. Finally, we point potential new users of TEM to resources to help launch their project.Transmission electron microscopy (TEM) has been an important technology in cell biology ever since it was first used in the early 1940s. The most frequently used TEM application in cell biology entails imaging stained thin sections of plastic-embedded cells by passage of an electron beam through the sample such that the beam will be absorbed and scattered, producing contrast and an image (see
TermDefinition
Beem capsulePlastic forms that hold samples in resin during polymerization
Blocks (bullets)Polymerized samples in plastic removed from the Beem capsule and ready to section
Block faceSmall surface trimmed on a block before sectioning
BoatWater reservoir in which sections float after being cut by a knife
CLEMCorrelative light and electron microscopy
DehydrationRemoval of water from a sample by replacement with solvent
Electron tomography (ET)A method to image thick sections (200–300 nm) and produce three-dimensional images
EmbeddingProcess of infiltrating the sample with resin
FixationSample preservation with low temperature and/or chemicals to maintain sample integrity
GridSmall metal support that holds the sections for viewing in the electron microscope
HPF/FSHigh-pressure freezing/freeze substitution sample preparation technique
Immuno-EMDetection of proteins in EM samples using antibodies
In-FXXKing credible!!!!Actual user quote in response to particularly beautiful sample. You may embellish with your own words.
KnifeA very sharp edge, either glass or diamond, used to slice off resin-embedded samples into sections
Pre-embedding labelingApplication of antibodies before fixation and embedding
Post-embedding labelingApplication of antibodies to sections on the grid
PoststainingStaining with heavy metals of sections on a grid
ResinLiquid form of the plastics used for embedding
RibbonCollection of serial sections placed on the grid
Serials sectionsOne-after-the-other thin sections in a ribbon
TEMTransmission electron microscopy
Thin sectionsThe 60- to 70-nm sections cut from the samples in blocks
TrimmingProcess of cutting away excess resin to create a block face
UltramicrotomeInstrument used to cut sections
Vitrification/vitreous iceUnordered ice in which samples can be viewed without fix or stain
Open in a separate windowTEM has proven valuable in the analysis of nearly every cellular component, including the cytoskeleton, membrane systems, organelles, and cilia, as well as specialized structures in differentiated cells, such as microvilli and the synaptonemal complex. There is simply no way to visualize the complexity of cells and see cellular structures without TEM. Despite its power, the use of TEM does have limitations. Among the limitations are the relatively small data set of cells that can be imaged in detail, the obligate use of fixed—therefore deceased—cells, and the ever-present potential for fixation and staining artifacts. However, many of these artifacts are well known and have been catalogued (e.g., Bozzola and Russell, 1999 ; Maunsbach and Afzelius, 1999) .A typical TEM experiment consists of two phases: the live-cell experiment, in which a cell type, possibly a mutant, is grown under given conditions for analysis, followed by preparation of the specimen and imaging by TEM. Specimen preparation for conventional TEM is comprehensively reviewed in Hayat (1970) and briefly described here (Figure 1).Open in a separate windowFIGURE 1:A brief flowchart showing the work to be done with different types of sample preparation for conventional electron microscopy (yellow background). The advanced cryo-EM techniques are shown with a blue background. For immuno-EM, the samples can be stained before embedding (pre-embedding staining) or the sections can be stained (post-embedding staining).  相似文献   

15.
Freeze-drying for electron microscopy     
RICE RV  KAESBERG P  STAHMANN MA 《Archives of biochemistry and biophysics》1955,59(2):332-340
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16.
Thrombocytes in electron microscopy     
ABDULLAEV GM  DUL'TSIN MS  TERENT'EVA EI  FAINSHCHTEIN FE 《Biulleten' eksperimental'no? biologii i meditsiny》1957,44(10):114-116
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17.
Microwaves for electron microscopy     
《Micron and Microscopica Acta》1990,21(4):217-222
Microwaves are electromagnetic waves with frequencies between 300 MHz and 300 GHz, corresponding to wavelengths between 1 m and 1 mm, respectively. Microwaves interact with a wide variety of materials. In fact, they can be used to heat dielectric materials. Diffusion and chemical-reaction rates are influenced by temperature increase. Many authors believe that, if microwave irradiation is optimally applied, the resulting microscopical images are of superior quality, because of good process control.In order to develop good microwave recipes for EM it is important to face the following questions:
  • 1.1. What is the influence of microwaves on the reagents?
  • 2.2. What are the basic mechanisms behind the procedure?
  • 3.3. What is the influence of temperature increase on the reaction rates?
  • 4.4. What is the optimal temperature?
  • 5.5. Does microwave irradiation cause destruction of, for instance, proteins or membranes?
  • 6.6. How to program the microwave oven? How does the load (container with reagent, if any, and specimen) influence the microwave irradiation? How to place the container in the oven?
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18.
Phase microscopy and electron microscopy of blood cells     
BESSIS M 《Biologie médicale》1957,46(3):239-288
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19.
Transmission electron microscopy of tissue prepared for scanning electron microscopy by ethanol-cryofracturing.     
W J Humphreys  B O Spurlock  J S Johnson 《Stain technology》1975,50(2):119-125
Tissue processed for scanning electron microscopy by ethanol-cryofracturing combined with critical point drying was embedded and sectioned for transmission electron microscopy. Study of specimens cut in a plane passing through the fracture edge indicated that preservation of cellular fine structure of fractured cells was excellent. Even at the most peripheral edge of the fracture there was no evidence that movement of cytoplasmic components occurred to distort the original structural organization of fractured cells. Lack of cytoplasmic detail in ethanol-cryofractographs has been due more to the nature of the fracturing of the tissue and to the obscuring effects of the metal coating than to structural deformation at the fracture edge or to limitations in resolving power of the scanning electron microscope used.  相似文献   

20.
Cryotechniques for electron microscopy: Comparison of freeze-fracture-replication method and cryoscanning electron microscopy     
T. Nei 《Cryobiology》1976,13(6):666-667
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