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Elucidation of the structure of biological macromolecules and larger assemblies has been essential to understanding the roles they play in living processes. Methods for three-dimensional structure determination of biological assemblies from images recorded in the electron microscope were therefore a key development. In his paper published in Philosophical Transactions B in 1971, Crowther described new computational procedures applied to the first three-dimensional reconstruction of an icosahedral virus from images of virus particles preserved in negative stain. The method for determining the relative orientation of randomly oriented particles and combining their images for reconstruction exploited the high symmetry of the virus particle. Computational methods for image analysis have since been extended to include biological assemblies without symmetry. Further experimental advances, combined with image analysis, have led to the method of cryomicroscopy, which is now used by structural biologists to study the structure and dynamics of biological machines and assemblies in atomic detail. This commentary was written to celebrate the 350th anniversary of the journal Philosophical Transactions of the Royal Society.
1. Introduction
Early members of the Royal Society, notably Robert Hooke and Antonie van Leeuwenhoek, invented new designs for the light microscope and applied them to imaging a previously unseen world of tiny creatures. Since then, the microscope has been one of the most powerful tools for extending the reach of the senses in both the life sciences and physical sciences. On a much smaller scale, the visualization of viruses, the causative agents of disease in humans, animals and plants, awaited the twentieth century development of the electron microscope, which can illuminate specimens with radiation of wavelength more than 100 000 times smaller than that of visible light. Viruses are assemblies that package their genetic code as nucleic acid, either DNA or RNA, for transfer from a host cell to their next target of infection. The electron microscope showed that while some types of viruses were variable in structure, others were remarkably uniform, either as rod-shaped or spherical particles. Structural studies of viruses and viral proteins by electron microscopy and X-ray crystallography have complemented biochemical and genetic approaches in understanding the life cycle of viruses, a subject important to both basic science and medicine.
R. A. Crowther's 'Procedures for three-dimensional reconstruction of spherical viruses by Fourier synthesis from electron micrographs' [1] described the first objective computational approach to determine the three-dimensional structure of a spherical virus particle from images. By combining electron images of identical, symmetric virus particles in different orientations, the paper describes the first 'single particle reconstruction', a term now applied to a method for structure determination of macromolecular assemblies. The paper was presented at a symposium on 'New developments in electron microscopy' at the Royal Society on 13 March 1970 organized by Hugh Huxley and Aaron Klug. These new developments were reported in three areas: (i) instrumentation, (ii) sample preparation, and (iii) image analysis. Early microscopy pioneers of the Royal Society would have emphasized progress in only the first two areas. Crowther's paper was a contribution in the third area of development and was an application of the new electronic computers of the era to extracting biological information from electron micrographs. In the subsequent 45 years, continued developments in these three areas have led to a general and widely applied method for structure determination of large macromolecular assemblies. Dramatic progress in the last couple of years has extended its application to atomic resolution studies of smaller asymmetric proteins and nucleic acid assemblies, providing biology with one of its most powerful tools for imaging molecular structure. It is therefore an instructive moment to consider the origins of single particle reconstruction as well as both the incremental steps and leaps that have brought electron microscopists and structural biologists to the present moment of excitement and optimism.
2. Electron microscopy and virus structure
The studies described by Tony Crowther in Philosophical Transactions and in related publications with colleagues were performed at the MRC Laboratory of Molecular Biology (LMB) in Cambridge, where during the course of his career he was a graduate student, research group leader and Joint Head of the Structural Studies Division. The LMB has an illustrious history of achievement in macromolecular studies including X-ray crystallography, the first method to reveal the atomic structure of proteins, and many of the developments in electron microscopy discussed in this essay happened there. In 1972, around the time of the spherical virus work, Crowther described the fast rotation function in X-ray crystallography, a procedure for finding the orientation of protein models in crystals [2]. Progress in X-ray crystallography and electron microscopy occurring at the same time and under the same roof reflects among other things the similar goals and underlying physical and mathematical principles of the two techniques.
Tomato bushy stunt virus (TBSV) is a small, spherical virus that has featured as a prototypical macromolecular assembly in several landmark studies in structural biology, including Crowther's first work on virus reconstruction [1,3,4]. In the electron microscope, the virus particles appear as highly uniform spheres with surface features suggesting polyhedral symmetry (figure 1a). Purified TBSV particles are so regular that they form well-ordered three-dimensional crystals with cubic-shaped unit cells implying that each virus particle is identical in structure. In 1956, Crick & Watson [5] summarized both structural and chemical evidence that spherical shells or 'capsids' of viruses such as TBSV contain multiple copies of a single kind of subunit with a regular packing, and because of this definite geometry, a plant virus could be considered a molecule. They argued that using many copies of a single protein to build the largest regular structure possible solves the problem of packaging the nucleic acid genome with a minimum of genetic information. The largest such regular structure is the icosahedron, a Platonic solid with 20 triangular faces and 60-fold symmetry. In the same issue of Nature, Don Caspar's X-ray photograph of a slice of the three-dimensional diffraction pattern of a TBSV crystal showed intensity spikes indicating an arrangement of fivefold, threefold and twofold symmetry axes consistent with the symmetry of the icosahedron [6].
During the 1960s Klug, Finch and co-workers studied the architecture of virus capsids by imaging viruses in the transmission electron microscope using negatively stained specimens [7]. In the negative stain method, biological specimens, normally found in an aqueous environment, are embedded in a thin layer of dried heavy metal salt, a preparation that is stable in the vacuum of the electron microscope. Because biological specimens are composed of light atoms, the structures appear in high contrast compared with the background stain, which scatters electrons strongly. This staining revealed the envelope of the particles at relatively low resolution, and though there could be distortions of the particle and other artefacts, the preparation method was adequate to see morphological features. These studies elaborated Caspar & Klug's theory of quasi-equivalence [8] which describes how capsids containing multiples of 60 subunits (e.g. 180 for TBSV) can be organized with icosahedral symmetry.
Their structural studies of viruses in stain also focused on a more detailed understanding of how the electron images were formed. By comparing images of virus particles that were sitting in stain on only one side versus those that were stained on both sides, they learned that images of virus particles uniformly embedded in stain were super-positions of the top and bottom of the capsid shells [9]. This is a consequence of the very large depth of field of electron lenses, which means that an electron image is a projection of the structure. By comparison, confocal light microscopes have nearly as good vertical resolution as in-plane resolution, so three-dimensional information about the specimen may be obtained by focusing at a series of planes through the specimen. In an electron micrograph, overlap of information from different levels in the specimen causes difficulty in interpreting the image. The images can therefore only be understood in terms of the three-dimensional structure that gives rise to the projections. The first approach to interpreting the images was to build three-dimensional physical models and make shadow projections of the models with light [10,11] and even more elaborate analogues for negative stain images [12].
Further insight into projection images in the electron microscope came from the analysis of rod-shaped viruses, such as tobacco mosaic virus (TMV), understood to be a helical organization of identical protein subunits that assemble around its RNA genome [5]. Owing to this periodic structure, an electron micrograph of an individual helical virus particle acts like a diffraction grating and laser diffraction was used as a method to analyse structure [13,14]. Subsequently, a new technology, a computer-controlled film densitometer (programmed by Crowther, then a graduate student) [15] was used to read the optical density at steps along an electron micrograph and store the data as a rectangular array of numbers (pixel values). The calculation of diffraction patterns could be performed on the digitized image in the computer by Fourier transformation, a mathematical technique in which an image or a volume is analysed in terms of its spatial frequencies. The Fourier transform of an image of a TBSV particle (figure 1b) shows strong intensities along the directions of the symmetry axes, consistent with Caspar's [6] observations.
Protein crystallographers had learned that the diffraction patterns recorded while illuminating different orientations of a crystal were portions of a single three-dimensional lattice or diffraction pattern. The three-dimensional diffraction pattern is related to the structure of the protein in the crystal through the mathematics of the Fourier transform. There is a direct mathematical or computational procedure to determine the structure of a molecule from its three-dimensional Fourier transform called Fourier inversion.
In the years immediately before Crowther's work on spherical viruses, a breakthrough occurred in the interpretation of projection images from the electron microscope through Fourier analysis, enabling the direct calculation of the three-dimensional structure or 'reconstruction' of the helical tail of T4 bacteriophage [16]. The interpretation of a projection image depends on the Fourier projection/section theorem: the Fourier transform of a two-dimensional projection of an object is equivalent to a central slice through the three-dimensional Fourier transform of the object. The Fourier transform of the T4 tail image is a single slice through the three-dimensional transform, but an important consequence of the helical symmetry is that all the slices about the helical axis are then known. Thus, for the special case of a helical specimen, an image of one particle is all that is required to calculate its three-dimensional structure, at least to low resolution. In concluding their work, DeRosier & Klug proposed a general principle for the reconstruction of non-helical particles in which data for all the required projected views would be recorded and combined [16].
3. Three-dimensional reconstruction of a spherical virus
At the time of the Royal Society meeting, Crowther et al. [4] had submitted an article to Nature describing reconstructions of both TBSV and human wart virus. The paper presented at the Royal Society meeting was a description of new methods for reconstruction with TBSV as an application. Crowther acknowledged Linda Amos, with whom he co-authored several papers at this time, for writing computer programs for the new procedures. Two approaches were used for recording the views required for reconstruction. In the case of the human wart virus, two views of each particle were obtained by tilting the specimen holder through a known angle, a forerunner of the multiple tilts used to collect data for tomography of non-symmetric specimens [17]. In the case of the TBSV particles, images of several different particles were used, with the stated advantage that individual particle imperfections would be averaged out.
The non-helical, spherical virus specimen presented a new problem. Images corresponding to different projections of the virus need to be combined in one reconstruction. This requires determining the orientation of different virus particles within the images and centring them in a single coordinate system. The specimen was a good candidate because the virus has distinguishable features in negative stain, and earlier evidence strongly indicated that it has icosahedral symmetry. A simple formula described the minimum number of views required to calculate a structure of a given diameter to a desired resolution. If an image could be assigned an orientation, then by application of the symmetry of the virus many more views of the virus could be calculated. Because the Fourier transform of an image represents a slice through the three-dimensional Fourier transform of the virus, then the three-dimensional Fourier transform can be filled up by a few images (five for the TBSV study), each with up to 59 symmetry-related views. The structure of the virus could then be calculated by an inverse Fourier transform as with crystals or helical specimens.
Thus, the key issue in the analysis of single particles is to assign the orientations. Crowther observed that any two slices of the three-dimensional Fourier transform have a geometric intersection through a line where each transform would have the same values, referred to as a 'common line'. In principle, the computer can search all the lines in two slices to find the common line, but this is very difficult for noisy, imperfect images. However, the symmetry of the virus can be exploited: one common line implies the existence of other common lines within a single image. A search for the number and location of many 'self-common lines' uniquely identifies the orientation of the icosahedral symmetry axes of the particle observed in the projection. The best assignment of a particle's orientation is one that gives the greatest numerical agreement between all common lines predicted for that orientation. Approximate values for the orientation and position of the particle in the image can be further improved by 'refinement'. Several refined images can thus be consistently oriented in the same coordinate system.
A number of significant computational issues needed to be solved to calculate the structure from separate projection images, including making sure that the data are sampled finely and regularly onto the final grid or volume on which the structure is calculated and presented. Some of the issues were discussed in an earlier work of Crowther et al. [18]. The method of calculation using a cylindrical polar coordinate system was an efficient solution with consideration of the available programs and computing power at the time. Modern programs that process all types of single particle specimens use a Cartesian coordinate system. Figure 2 shows Crowther's handwritten flowchart for the procedure.
The map of the virus calculated in this way (figure 3a) showed how 90 morphological units, each explained as a dimer of the 180 capsid monomers in the virus, were arranged with respect to the icosahedral symmetry elements as well as local symmetry elements. The paper contained, for the first time, several recognizably modern demonstrations of the success or reliability of the reconstructions. Calculated projections of the reconstruction agreed with the experimental images (figure 3b). The paper gives a measure of the reconstruction quality (the agreement between matched common lines) as a function of resolution and showed a correlation to the expected resolution limit of negative stain (approx. 30 Å). This is sufficient to confirm the assumption of icosahedral symmetry and that the alignment methods are working. The modern expert will also recognize a number of other familiar features such as the enforcement of a consistent absolute hand and magnification as determined from the 'cross common lines' between particles.
In short, the paper had accomplished three-dimensional image reconstruction from two-dimensional images of non-crystalline single particles. Spherical viruses are an ideal model system for developing methods, and though not the most general specimen, they are an important generalization from helical or crystalline specimens. Because of the limitations of the large grain size of negative stain and the small number of images, the structure of TBSV could only be determined to very low resolution. The paper was an important beginning to the single particle reconstruction field and a glimpse of the road ahead. While Fourier-based methods have been widely developed and applied, alternative iterative approaches to structure determination from projections in real space [19,20] have proved useful in some contexts. Fourier methods are also the natural way to treat microscope aberrations, an essential part of high-resolution image analysis and the subject of another paper presented at the meeting in 1970 [21]. In addition to Crowther's original programs, modern variations of the procedure are used by those active in structure determination of icosahedral viruses [22–24]. The continued relevance of the methods, at least in outline, and the clarity of their presentation make Crowther's paper worthwhile reading for a student today.
The atomic model for TBSV, the first high-resolution view of a crystalline virus, was determined at 2.9 Å by Stephen Harrison and co-workers using X-ray crystallography [25] (figure 3c). The structure showed how the capsid protein uses two flexibly linked domains to adopt the different conformations required by quasi-equivalent packing. Radiation-sensitive virus crystals, many weak reflections and the difficulty of solving the crystallographic phase problem for a large molecular assembly posed huge technical challenges and led to important new methods in crystallography. Yet, the symmetry of the virus posed constraints on the density that were used to solve the structure. The properties of TBSV, its regularity and symmetry, were exploited in both electron and X-ray studies. Thus, TBSV and other spherical viruses helped to drive the development of ideas and methods in both fields [26–28]. In the two subsequent decades, X-ray crystallography remained the widely applied high-resolution technique while electron microscopy was applied to lower-resolution studies of large assemblies. Very few would have thought that eventually electron microscopy could routinely reach atomic resolution.
4. The development of high-resolution single particle electron cryomicroscopy
Progress in the field of three-dimensional electron microscopy resulted from many developments in each of the three areas described at the 1970 Royal Society meeting. As anticipated in the conclusion of Crowther's paper, structure determination from individual particle images could be extended to particles with lower symmetry or even to proteins with no symmetry at all. For an asymmetric particle, many more views are required and the specific methods employed by Crowther for determining orientations could not be applied. Furthermore, images of particles in negative stain consist of desirable signal from the particle as well as noise that comes from the underlying structure of the carbon film support, particle distortion, stain variation and the photographic process. But by averaging similar views of a particle, the common signal of the particle in the images is increased compared with that of the noise, resulting in a higher signal-to-noise image. The ribosome, a large asymmetric macromolecular assembly of central biological importance as the engine of protein synthesis, was an excellent target for electron microscopy and drove the development of methods for studying asymmetric particles. The key developments included methods for statistical classification and alignment of similar views of the particles so that many high signal-to-noise averages of the particle can be obtained [29]. In addition, a method for determining the relative orientations of the views of an asymmetric specimen for subsequent three-dimensional map calculation was required. The first successful strategy was a two exposure method called 'random conical tilt', which assigns orientations to particles in an image field recorded when the specimen is tilted, provided that the same particles all display a similar view to each other when the specimen is untilted (usually due to a preferential interaction of the particle with the carbon) [30]. A computational procedure called angular reconstitution for aligning images based on real space common line projections with similarities to Crowther's Fourier transform methods also succeeds in determining the relative orientation of high signal-to-noise particle views but without requiring tilting of the specimen [31]. A map achieved by either method can be used to calculate projections that can then be matched against experimental views, thus assigning the experimental view an orientation. An iterative process of model-based refinement improves the assignment of orientations and the quality and resolution of the resulting map. These methods were later applicable to the frozen-hydrated specimens studied by electron cryomicroscopy (cryoEM) [32]. Progress on negative stain specimens, however, was destined to reach a limit, providing only low-resolution molecular envelopes. New experimental approaches were required for studies at high resolution such as those obtained by X-ray crystallography.
High-resolution structure determination by electron microscopy was first demonstrated for bacteriorhodopsin (BR), the light-driven proton pump that forms crystalline patches in the purple membrane of Halobacterium halobium. In 1975, Henderson & Unwin [33] calculated a three-dimensional map at 7 Å resolution from two-dimensional BR crystals showing the trans-membrane α-helices of a membrane protein for the first time. A two-dimensional crystal is a periodic array of molecules one molecule thick in which each is held in fixed position and orientation as determined by unit cell dimensions and symmetry of the crystal. Different views of the molecules, each corresponding to a slice through the three-dimensional Fourier transform are obtained by tilting the specimen. Fourier transforms of their images reveal a crystalline lattice, and their study is sometimes called electron crystallography, but the images provide both amplitudes and phases, unlike X-ray diffraction patterns, which give only amplitudes.
The work reported several major methodological advances. They studied unstained specimens in glucose, which effectively replaced the water in the sample but preserved native protein structure to high resolution. The resulting maps of the protein show density variations in the structure of the protein itself, including its interior, rather than the stain distribution. Such thin biological specimens are composed of light atoms and require small amounts of defocus during imaging to enhance their contrast, typically between a few hundred nanometres and up to a few micrometres. Furthermore, biological specimens without stain are extremely sensitive to irradiation by the electron beam, more so than the negative stain preparations discussed so far. The fading of the crystalline patterns of BR showed that images should be recorded with less than five electrons impinging on a square Ångström of the specimen to preserve high-resolution features. These studies therefore employed imaging methods that minimized the exposure of each crystalline specimen to a single 'low dose' image. For low dose images, statistical variation in electron counting is a major source of the noise. The diffraction patterns calculated from the images are the computed average of the scattering of all the molecules in the crystal. Analysis of the patterns is similar to averaging images of all the individual molecules in the crystal, thus overcoming statistical noise and giving a high-resolution image of the molecule.
In subsequent years, Henderson and colleagues developed experimental and computational methods for analysing two-dimensional crystalline specimens to high resolution, ultimately obtaining a map for BR at 3.5 Å resolution, sufficient to build an atomic model [34]. Data collection on specimens held at liquid nitrogen temperature (−190°C) is critical for reducing radiation damage and recording high-resolution images. Important instrument developments during this time were computer control of the microscope for low dose image acquisition and field emission gun microscopes which provide a more coherent source of radiation than earlier thermionic electron guns [35]. Coherence is critical for recording high-resolution information and for calculating the effect of microscope aberrations and the focal setting in the image, parameters that are necessary for extracting high-resolution structural data from electron micrographs. Although near atomic resolution structures have been demonstrated for several other two-dimensional crystals, they remain a specialized specimen type, powerful for membrane proteins, but were important for developing experimental and computational methods and showing what was possible with electrons.
The breakthrough in sample preparation described by Jacques Dubochet and co-workers, following earlier attempts to prepare frozen-hydrated specimens [36], provides a general method for sample preparation that preserves structure to high resolution [37]. This could be applied to a wide range of specimen types for imaging by 'cryoEM' from single particle specimens to whole cells. A thin film of the specimen in buffer is plunged into a cryogen, such as liquid ethane, cooling it rapidly before ice crystals can form, and creating the near native environment of amorphous ice. The thin film remains stable in the microscope vacuum when held at liquid nitrogen temperature. A wide range of specimens including Semliki Forest virus [38,39] were frozen in random orientations in thin films over holes, avoiding the granular noise contributed by the carbon support. When low dose procedures are applied to such specimens, the resulting images are noisy, but have the aforementioned advantages of an unstained specimen. A single high-resolution image of a field of identical particles in random orientations can be recorded. However, averaging of different identical particles in the same orientation reinforces the signal over the noise, creating higher contrast views of the particle for reconstruction. By applying image processing methods such as described for the ribosome, a wide range of specimens were studied, but mostly at a resolution worse than 10 Å.
The near atomic resolution information obtained from two-dimensional crystals and some helical specimens showed that high-resolution protein structure determination was possible with electrons, and the number of molecules in the crystalline lattice gave an indication of how many images of individual, unstained protein molecules would be required to reach atomic resolution. However, the molecules in crystals have the benefit of being aligned to each other, and their orientation is known from the easily measurable orientation of the lattice. In single particle analysis, each molecule's position and orientation must be determined in the image. The ability to find the orientation of a particle depends on whether it is large enough to scatter electrons strongly compared with the background noise, i.e. how visible it is. A theoretical calculation showed that for images limited only by the noise associated with low electron dose, 10 000 particle images should be sufficient to reach atomic resolution (3 Å) for a single particle of any size [40], though the lower size limit was approximately 100 kDa. At the same time, quantitative examination of the amount of contrast in cryoimages showed that they were imperfect due to a number of factors including movement of the specimen, sometimes induced by the electron beam itself. Movement of the specimen effectively blurs the image and limits the higher-resolution information [41]. The consequence of image imperfections is that many more particles are required to reach high resolution, even for a relatively large complex. Perhaps even millions. And smaller objects cannot be aligned reliably at all. The calculation stimulated efforts to reach high resolution by showing that relatively few particles would be required if some of the experimental and theoretical problems could be solved.
Instruments, sample preparation and image analysis were now poised for high-resolution studies of single particles. Symmetric viruses were the most likely to get there first because they are highly visible in amorphous ice due to their large size and are therefore easier to align. Aligning and averaging icosahedral particles includes 60 times more monomers or 'asymmetric units' than for an asymmetric particle. An important milestone was achieved by imaging a virus sub-particle, the icosahedral capsid assembly of hepatitis B virus, published in Nature by two groups [42,43]. In the study by Böttcher & Crowther, 6400 virus particles were averaged (384 000 asymmetric units when 60-fold symmetry is taken into account) to obtain a three-dimensional map at 7.5 Å resolution. The map revealed the fold of the polypeptide chain in the monomer and allowed the approximate assignment of amino acids positions, the first time this had been achieved for a non-crystalline specimen. This was a big leap from the five particles presented in Crowther's original description of the methods. Common lines were again the basis for image analysis of the particle, but this study included 'cross common lines' as a means of matching projections of a model with images [23]. The calculations required a huge amount of computer time for the refinement of particle orientations and correction for microscope aberrations necessary to bring out small details of the structure. About 10 years later, the high-resolution structures of the rotavirus double-layered capsid particle (50 MDa) [44] shown in figure 4 and the infectious sub-virion of aquarevious (72 MDa) [45] presented icosahedral particle maps that were interpretable by atomic models. This means the trace of the polypeptide chain through the map could position the amino side chain positions with certainty. These maps required about 10 000 icosahedral particles. Maps of smaller particles were improving but they did not approach atomic resolution.
For many years, single particle images have been recorded on photographic film, which captures high-resolution detail, and, for example, was the recording medium in the atomic resolution virus studies mentioned above. When electronic detectors (phosphor-based CCD cameras) became available, they were used widely because of their speed and convenience, even though film was a better detector. In the last couple of years, a new generation of direct electron detectors (DDD's or Direct Detector Devices) has been applied to imaging single particles, producing better images than film [46]. In addition to improving image quality at high resolution, the new detectors can record many images per second, thus capturing the image of a particle over many frames, recording a movie of the specimen. These revealed the movement of the specimen during the course of a low dose image. Suddenly, a new opportunity for image analysis arose. By computationally aligning the particles between frames, a new image that is corrected for the movements is obtained [47–49]. In addition, the frames that contain high-resolution data can be chosen for analysis, and the frames where the specimen shows damage discarded. The new structures calculated from these movies showed dramatically improved high-resolution features and a 'resolution revolution' was proclaimed [50].
The asymmetric 1.9 MDa 54S subunit of the ribosome, much smaller than the viruses described at high resolution, could now be solved close to 3 Å by electron microscopy and from fewer particles than previously imagined [51] as shown in figure 5. This resolution improvement is critical because, as learned from years of X-ray analysis, it is then possible to extend from a secondary structure model to an accurate placement of amino acid side chains or nucleotide bases, positioning the chemical groups that are the basis for the molecular mechanism. The tetrameric TRPV1 ion channel, a 300 kDa membrane protein complex, was determined to 3.5 Å in some regions [52]. The current state of the field is represented by the 4.5 Å structure of γ-secretase, a membrane protease that is responsible for the aberrant products that form amyloids associated with Alzheimer's disease [53]. Though achieving slightly lower resolution than the other studies, γ-secretase is an asymmetric molecule of about 170 kDa. While cryomicroscopists previously chose projects that were suited to the technique, there are now manifold specimens in this lower molecular weight range for which microscopy will be a fair competitor to crystallography.
5. High-resolution structure determination by electron cryomicroscopy: the future
It would appear at the time of writing that many of the major obstacles to the atomic resolution imaging of non-crystalline biological specimens by cryoEM have now been surmounted, some of them in the last year or so. Electron microscopy is a technique with general applicability to a range of specimen types, including single particles, fibres and two-dimensional crystals. One hundred years after Bragg's Nobel Prize for the development of X-ray diffraction analysis, single particle cryoEM joins X-ray crystallography and NMR spectroscopy as a high-resolution structural method for biological macromolecules. Because of its similar goal to X-ray crystallography, the single particle approach has been described as crystallization in silico because the alignment and averaging of images is done by the computer and no longer requires the growth of well-ordered crystals of the specimen. Microscopists have not yet reached atomic resolution for the lowest molecular weight limit or smaller number of particles predicted by theory [40], but avenues for improvement have been identified and may push the method toward much lower molecular weight particles. These improvements may include new detectors that count individual electrons and specimen supports that reduce movement.
Future developments of three-dimensional electron microscopy with impact in biology will include further generalization of the types of specimens that can be imaged at high resolution. Even with recent rapid advances, high-resolution single particle analysis is largely restricted to proteins that are biochemically purified and are assumed to be nearly identical in structure. In the future, electron microscopy will include many more investigations where these assumptions can be relaxed to include specimens with structural flexibility and heterogeneity. The function of many molecular machines is achieved through large structural transformations. The classical approach is to trap the molecules in a well-populated intermediate for imaging. But by breaking the molecular machines out of crystalline lattices, we can hope to image the structural transformations happening in solution. Examples include the membrane rotary ATPases, whose motion is linked to the synthesis or hydrolysis of ATP [54], and the chaperonin GroEL/ES [55], which assists the folding of proteins.
Of course, when a specimen has conformational or compositional heterogeneity, it is more complex to tell a computer how to classify and average particle images. Any particle image can be assigned to one of several different structures and to different orientations. Each member of the ensemble of structures requires an independent structure determination and many more particle images are required for the whole multiple structure dataset. Statistical approaches to image processing such as maximum likelihood have contributed to the resolution revolution [56], but at the same time are probably in their infancy. Better quality images will always help when dealing with problems of increasing structural complexity as will large datasets from automated data collection, but we shall have greater challenges in analysing them. As cryoEM becomes more useful, it will also become a technique applied by the non-specialist, in which case it may be the computer that arrives automatically at the answer to a complex problem. Therefore, computational methods that can be applied reliably will require continued development. Crowther and co-workers in several ways addressed the question of whether their TBSV structure was correct. Validation methods [57–59] that prevent computers from refining an incorrect model against noisy data [60] must continue to be developed as cryoEM is applied to the most challenging specimens.
While symmetrical viruses can be solved by single particle methods, many viruses that are important human pathogens are variable in structure or pleomorphic, such as the lipid-enveloped influenza viruses. Each virus is different, and images of different viruses cannot be averaged together, and they are best studied by cryotomography. In tomography, all the unique projection images required to determine the structure by electron microscopy are recorded from a single specimen as a tilt series, and the resulting three-dimensional reconstruction is called a tomogram [61]. There is an advantage over single particle reconstruction in that the relative angles of the images are known from the experiment and they do not require de novo alignment. However, the single specimen receives a larger electron dose, and accumulated radiation damage places a limit on the resolution of the resulting structure. Nevertheless, the low-resolution maps obtained by cryotomography of influenza virus [62] may be interpreted, for example, with high-resolution X-ray models for the surface glycoproteins, the haemagglutinin [63] and the neuraminidase [64], creating pseudo-atomic models of the virus. Higher-resolution molecular structures are possible through tomography by aligning and averaging tomogram sub-volumes containing identical or similar molecules. Sub-tomogram averaging for a helically ordered specimen has reached sub-nanometre resolution [65], an important threshold for visualizing the fold of a protein. On a larger scale, one of the most exciting endeavours is the high-resolution structural study of cells by cryotomography with the goal of imaging proteins and their assemblies in vivo [66].
Where molecular averaging is not possible, there may still be opportunities for higher-resolution studies through improved instruments and improved image analysis. The defocus mechanism of contrast used in cryoEM at present is imperfect because the contrast is not uniform across all resolution ranges. Zernike's phase plate for the light microscope had been proposed by 1940 and has been used in light microscopy for many years. Phase plates for electron microscopy are currently an active area of research and could help record high-contrast, in-focus images. Interestingly, the Transactions published a paper presented by Nigel Unwin at the 1970 Royal Society meeting that contained a description of a phase plate consisting of a thin spider web coated with metal suspended across the objective aperture of an electron microscope, and this produced phase contrast images of TMV [67]. Though better imaging methods and calculations may allow us to get more information for a given electron dose, radiation damage to the specimen is ultimately the limiting factor for imaging with electrons and X-rays. It is a question for the more distant future if there are approaches to atomic resolution imaging of radiation-sensitive biological specimens that can beat the radiation damage problem. One proposal is to record images or diffraction patterns extremely rapidly (within femtoseconds) with a pulsed radiation source so that the structure of the specimen is recorded before it can react to the damage [68].
There has been a history of improvement in the instrumentation used to image biological macromolecules while the means to record the image and analyse its features has similarly undergone dramatic changes. Robert Hooke's spectacular drawings in Micrographia tell us what he saw through the eyepiece. The development of camera technology and computational methods in microscopy has allowed a more detailed and objective analysis of the specimen. In electron microscopy, image analysis revealed molecular structure by first exploiting the special features of crystalline and helical specimens and then finding ways to analyse less periodic specimens, as exemplified by Crowther's paper. Advances with prototypical specimens revealed how instrumentation, sample preparation, data collection and analysis could be improved to solve the structure of the most general, asymmetric specimens. Our ability to collect vast image datasets and analyse them rapidly by computer will no doubt extend our understanding of biological structure and function. Images are now just frames in a movie of the specimen's lifetime. Computational image analysis has become an essential lens through which we observe and attempt to understand the movies of the asymmetric and dynamic structures we call life.
Funding statement
P.B.R. is funded by the Medical Research Council (UK) under programme code U117581334.
Author profile
Peter Rosenthal studied physics at Harvard College and then obtained a PhD in the Harvard biophysics programme working on X-ray crystallography of influenza virus surface glycoproteins. He moved to the MRC Laboratory of Molecular Biology in Cambridge to work on the development of experimental and computational methods for high-resolution structure determination by single particle electron microscopy without crystals. Since 2005, he has been a Programme Leader in the Division of Physical Biochemistry at the MRC National Institute for Medical Research at Mill Hill, London. His research applies electron microscopy to the study of biological structure from the molecular to the cellular scale including the structural analysis of lipid-enveloped viruses. In 2015, his laboratory will join the Francis Crick Institute in London.
I thank Tony Crowther for discussions and material and Justin Molloy, John Rubinstein and Michael Vorenberg for comments on the manuscript.
One contribution of 18 to a theme issue 'Celebrating 350 years of Philosophical Transactions: life sciences papers'.
The featured article can be viewed at http://dx.doi.org/10.1098/rstb.1971.0054.
© 2015 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited.


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Soulja Boy Fires Shots At 50 Cent Again
Soulja Boy dissed 50 Cent on Twitter:
Shoutout to 50 for the free promo. Stupid ass nigga 😂 anybody can break in a house when nobody is there. ill shoot a nigga for my respect— Soulja Boy Tell 'Em (@souljaboy) January 13, 2017
Amid news that his Hollywood Hills crib got burglarized, Soulja Boy got the Twitter fingers Friday and fired shots at 50 Cent. Apparently, Soulja did not take too kindly to Fif posting a photo on Instagram making fun of the burglary. Over $20K worth of values was stolen. To make matters worse, the landlord said he'll take legal action unless the social media warrior pays up his late rent.
The "Crank That" rapper went on a mini rant before boarding a flight. He even mentioned that he had a new draco. In 2008, he shot at a group of burglars in his home, killing one. He recounted the story to DJ Vlad inspiring the #SouljaBoyChallenge.
He is currently training for his upcoming boxing match with Chris Brown. 50 Cent and Mike Tyson are training C-Breezy while Floyd Mayweather, Jr. is mentoring the "I Got That Sauce" emcee.

subset: commoncrawl


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Watch Zero
Genres: drama
Plot Summary: In a world that judges people by their number, Zero faces constant prejudice and persecution. He walks a lonely path until a chance encounter changes his life forever: he meets a female zero. Together they prove that through determination, courage, and love, nothing can be truly something.
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Zero Video Links:
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Zero English Dubbed (Youtube)
*After watched Zero, feel free to rate it, social bookmark or leave review comments about it. It will help our site become better and others member easily on choosing good anime series. Thanks for your contribution !!!
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*Tags: watch Zero, english dub, download Zero, stream Zero online, episode sub, animated film


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New Popular Hobby Mechanics & Electronics
When I was growing up, I loved hobby technology. From model rockets to personal computers, from CB radio and electronics kits to chemistry sets and garage fabrication, I was always building, breaking or experimenting with something... and frequently combining whacky ideas from different domains.
In those days, Popular Mechanics and Popular Electronics magazines would feature new technologies - working or still in the labs - as well as projects to build. They were a good influence. I was sad to see that much that kind of thing seemed to fade in popularity in the 90's and 00's, for whatever reason.
Now, I see the popularity returning, and a good thing, too - America needs engineers and technology innovators now more than ever. Plus, it's just great fun!
This page is dedicated to 21st-century items I find around the net that remind me of that hobby-technology, learning and experimentation mode of learning. Of course, everything is a generation or two more advanced than when I was a kid, so I must admit, some of this stuff just baffles or amazes me. Which, as I recall from my youth, is much of the point. :)
Newest finds are at the top of the list:
The ArduCopter, a remote-controlled, programmable-flight flying device with a hi-resolution video camera, GPS and more on-board.
Make Magazine and their YouTube channel belong here, even though I have them listed in the sidebar.


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Choose from a variety of Light Box inserts for our Inspirational Accent Lamp.
"Having a place to go is Home
Having someone to love is Family
Having both is a blessing."
Each light box and quote insert is sold separately.
The light box insert measures 9 3/4" x 7 1/2".
See ALL Inspirational Light Boxes and Inserts.
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Let's cut to the chase. We all need traffic to our sites. That's a given. You do, I do, we all do. Even the SEO providers need it! But most cannot afford that astronomical fees that many Search Engine Optimization companies charge. And then, what guarantee do you have that you'll get what you pay for? None. Of course, they'll tell you a story that "the work is already done so it's difficult to refund for work done."
You could do it yourself. That's an option but a poor one, and here's why. Google, the number one search engine out there, changes their rules significantly about every year. It throws the online marketers into a scramble to see what changed and how to build up their rankings again. You as a business professional are good in the medical or construction or services industry. If you weren't you wouldn't be in business. Just like it took you years to learn and be who you are, so it is with the online marketing crowd, myself included. Sure, you may get lucky and rank in the search engines for the term "laying a hand-crafted metal roof in Spokane Washington" but who is searching for that term?
That's why it pays to hire professionals to do that work for you. But the dirty little secret is this, if you are a small business with anywhere from 2-10 employees and sales well under a million per year, you probably do not need the services the big boys charge. If you're in that category and paying more than $300 per month for SEO services, it's too much. That's our "sweet spot". We thrive on ranking businesses such as yours. Why? Because that seems to be where businesses need the most help.
Now, of course you can pay more to rank more keywords and we do acknowledge the need for that but when you're just getting started and getting into the entire online marketing, you should be fine for a sub-300 price range.
The picture at left illustrates the exuberance at getting the SEO services at a modest price. Ok. A little cheesy, but hey.


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SOURCE: Source Financial Inc.
October 23, 2013 08:21 ET
Moneytech Produces Game-Changing Payment Integration Gateway With Leading eCommerce Solution Provider
Moneytech Produces Game-Changing Payment Integration Gateway With Leading eCommerce Solution Provider
LOS ANGELES, CA--(Marketwired - Oct 23, 2013) - Source Financial, Inc. (OTCQB: SRCF) ("Source"), an innovative financial services and solutions provider, announces today that its subsidiary Moneytech LTD ("Moneytech"), a leading Australian financial solutions provider, has teamed up with VARCentral to integrate its payment gateway with the VARCentral eCommerce Solution for delivery of improved and seamless IT reseller solutions.
By combining Moneytech's payment gateway with the VARCentral eCommerce Solution, IT resellers can access product pricing and availability between a vast range of distributors, but with the convenience of managing one account for many distributors, which facilitates quicker ordering and payment processing. This can efficiently overhaul their processing and management practices, thus saving the user time and money. Resellers will know their exact available funds in real time and are able to then allocate those funds, so, if desired, they have the ability to pay any distributor, automatically, at the point of raising the purchase order in the VARCentral system.
The companies' payment gateway highlights their commitment to improve their client's results by delivering cost savings and efficiencies. VARCentral's innovative solutions, which are powered by ePages (one of the world's leading platforms that has over 75,000 online merchants worldwide) are available to a network of IT resellers whom will now have access to Moneytech's credit facilities, allowing them to purchase inventory with flexible interest free terms and credit limits up to $250,000.
"Moneytech's collaboration with VARCentral showcases a powerful combination of financial processing and IT reseller solutions that's birthed an amazing cross-sector innovation platform. This thrilling technology underlines the amazing possibilities that can be created by working together to maximize the potential of our combined strengths. This is a technology that can transform the way e-retailers do business, as we improve their supply chains, increase the range of product available to them and save them money on the bottom line, and I look forward to sharing our vision with the online reseller community," stated Hugh Evans, President and CEO of Source.
"This collaborative effort created the right product at the right time and it's a huge step to improving the reseller business model. Today's marketplace tends to reward the most agile players and this is an idea that can revolutionize the industry and can seamlessly be converted into practical solutions that users will reap tremendous benefits from," said Michael Blumentals, Managing Director of VARCentral.
About VARCentral:
VARCentral is powered by ePages, one of the world's leading platforms with 75,000+ online merchants world-wide. Tailored precisely to fit the needs of Australian IT Resellers, VARCentral provides an eCommerce solution that is not only affordable, but also gives you the necessary tools to successfully grow your IT business.
For further information on VARCentral please visit www.varcentral.com.au, or www.demo.varcentral.com.au for a demo.
About Source Financial:
Source Financial, Inc., through its two subsidiaries Moneytech Limited and WikiTechnologies, offers a complete suite of financial products for Consumers and Businesses.
Moneytech offers four main products to the Australian business community : 1) Credit Express - trade finance facilities for SMEs of $25,000 - $1 million, 2) Confirmed Capital - 100% Accounts Receivables funding for SMEs up to $5 million, 3) Card Solutions - Wholesale gift and prepaid debit card solutions, and 4) Foreign Exchange solutions - Spot Transactions, Forward Cover, Derivatives and Options. Moneytech's targeted financings are $3-$5 million dollars.
The Company's website is located at: www.moneytech.com.au
WikiPay, a Money Service Business, is a low-cost, mobile and online money transfer and payment solution operating through all 50 United States. For more information, please visit: www.wikipay.com.
WikiLoan is a low-cost peer-to-peer lending platform that works through the participation and collaboration of family and friends through social networks. The company's website www.wikiloan.com provides repayment schedules and documentation for loans, along with proprietary administrative tools, which enable users to securely pull credit reports and automate the loan repayment process.
FORWARD LOOKING STATEMENTS This news release may include "forward-looking statements" regarding Source Financial, and its subsidiaries, business and project plans. Such forward looking statements are within the meaning of Section 27A of the Securities Act of 1933, as amended, and section 21E of the United States Securities and Exchange Act of 1934, as amended, and are intended to be covered by the safe harbor created by such sections. Where Source Financial expresses or implies an expectation or belief as to future events or results, such expectation or belief is believed to have a reasonable basis. However, forward-looking statements are subject to risks, uncertainties and other factors, which could cause actual results to differ materially from future results expressed, projected or implied by such forward-looking statements. Source Financial does not undertake any obligation to update any forward-looking statement, except as required under applicable law.
Investors may contact: Market Street Investor Relations Inc. at (877) 218-9173
Investors may contact:
Market Street Investor Relations Inc.
(877) 218-9173
Investors may contact:
Market Street Investor Relations Inc.
(877) 218-9173
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Sanctuary DMV trains and coordinates volunteers to accompany you to your immigration or court appointments, hearings, and check-ins.
These volunteers are present as witnesses and support during the stressful process of navigating our unjust immigration system. In an emergency or if an individual is detained, accompaniers can relay the information to the family and/or friends and alert networks of support.
Accompaniers cannot provide legal counsel and are not present to legally represent those they accompany.
See this flyer to connect with accompaniment support.


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So this happened:
I graduated! We got this non-slip yoga mat during the ceremony… CorePower's way of saying 'good job!' There were tons of smiles, lots and lots and LOTS of tears. I cried off all my makeup. Dammit. But I was kinda cute before the waterworks started. Observe:
So the training has wreaked ALL KINDS OF HAVOC on my skin. Not to worry, I promise you that I'm setting this madness straight with a peel. Maybe this weekend. My skin is so rude. Just cystic pimples and spots… THE SPOTS. Friggin' hyperpigmentation. You know what? Not cute. I promise all will be well soon. Hmph.
In the meantime, I used Tom Ford Traceless Perfecting Foundation to even out the madness. That stuff is GENIUS. My color is Caramel (thank you to my girl Gabi for matching me sight unseen!). There was no time on Earth to go to a store and look at it so I had Gabi just take a look, and pick out my color. Stuff's PERFECT. So weightless, and very easily buildable (without looking like you built anything). Ya'll have no idea how many spots I had to deal with. And the brows… oh the brows. My tech was sick the day before graduation, so I had to see about making them work myself. I know… #lagirlproblems. So raggedy. But lookit, this was a big deal and I wanted to look my best.
Teacher Training kicked my butt, wrung me out, and hung me up to dry. But I'm a million times better for it. It has changed everything about me that I thought I knew, and had made me so excited for the journey ahead. I'm going for Extensions (advanced training) in a few weeks. If ya'll thought I was frazzled now…
This time, I'm keeping a peel handy (I haven't used it in so long that I lost it and had to order another bottle) to take care of this skin. I love the foundation, But I really want to wear less of it, so that more of me can truly shine through.
12 comments
Congratulations!! You are such an inspirational person. YOU SAID you wanted to do this and YOU MADE it happen. I don't know you like that but I feel so proud for you. Best luck and many blessings on the rest of your journey. And you look gorgeous so don't even worry about those "spots".
Congratulations Sha!!! This is such a wonderfully huge accomplishment! May the world of Holistic Wellness continue to recieve you with open hearts, bodies, and minds!!! You are a PHENOMINAL WOMAN!!!
Congrats! Your skin looks great despite the problems that you've had. I'm going through the same darn thing. I've got pimples in places where I never breakout (my cheeks and forehead). Now I need full coverage foundation 😦
I really want to try that TF foundation but doubt that my budget will allow it.
I'll give that a try tomorrow. One of the spots is just under my cheekbone and no matter what I do to conceal the spot, usually dab Nars creamy concealer on it, my blush brush wipes it away. Today, I didn't put on blush and you can't see the spot.
Have a good weekend!
Woohoo! Congratulations to you. You have been working SO hard.
[…] then I decided to become a yoga teacher, and I started dreaming in color. Suddenly I wanted to marry all manner of blues, greens, pinks, […]


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Stafford County (Va.)
Follow Marlborough Point Road down to the eastern tip of Stafford County, and you will pass by lots of new housing mushrooming into the forests and fields that were once favored by both the Native Americans and colonial settlers. This section of the county is home to not just centuries of local history but millennia.
"Now the temp'rance army's marching,
Wives and sisters in the throng,
Shouting 'Total Prohibition'
As we bravely march along!" - from the Temperance Army song
Did you know it was never illegal to drink during Prohibition? The 18th Amendment to the Constitution, also known as the Volstead Act, made the production, sale, and transport of intoxicating beverages illegal but said nothing about actually drinking the stuff. It contained some exceptions, too. For example, a doctor could prescribe medicinal whiskey to his patients. The production and distribution of liquor, once handled by legitimate businesses, became the province of criminal gangs. Can you say Al Capone? Respectable folk patronized illegal speakeasies. New York City alone had an estimated 30,000 speakeasies! As organized crime grew, and drinking gained more social acceptance, support for Prohibition waned, and in 1933 the 21st Amendment repealed the 18th.
Virginia was ahead of its time, banning alcoholic drinks more than three years before national prohibition was enacted in 1920. Learn more about this fascinating period in Virginia's history, including the long-lasting effects still felt today. With support from the National Alcoholic Beverage Control Association, the Virginia Alcoholic Beverage Control, and the Virginia Distillers Association, Howell Branch will host the Library of Virginia's exhibit, "Teetotalers & Moonshiners: Prohibition in Virginia, Distilled," June 11 - July 21, during regular library operating hours.
By Janet Payne
Janet Payne is the retired fine arts coordinator of the Stafford (VA) County Public Schools. This article originally appeared in the International Review of African American Art, volume 16, number 1, and is reproduced here with the permission of this publication.
In 1996 on one of my many visits to the Hampton University Museum, I had the opportunity to see the recently acquired Countee Cullen collection. As I viewed the familiar names of African American artists, I noticed an artist unknown to me—Palmer C. Hayden of Wide Water, Virginia. Could that be the same Widewater in Stafford County where I am the fine arts coordinator? How could this be? My research on the Stafford-born artist Palmer C. Hayden began in this moment.
Local Yearbooks in the Library
Check these links for yearbooks we hold in the library:
The Porter Branch is getting an exciting new look! Stafford County has contracted for "bookstore"-style shelving for the adult and teen collections, to be installed in July. These wooden shelves are lower and more attractive, allowing materials to be more accessible. Customers will no longer need to climb on a step stool to reach the top shelves! These shelves also provide increased visibility throughout the branch and will allow more natural light to shine through from the beautiful windows at the far end of the building.
There have been newspapers published in
Greg Riddlemoser, Director of Elections and General Registrar for Stafford County, has elected to share his reading choices with the Central Rappahannock Regional Library community:
Making reading recommendations is always dicey. We like what we like, and we benefit from our reading in our own unique ways. I guess that is one of the things that make books soooo powerful. I offer below a smattering of the stuff I like from light to heavy.
Every year brings a lot of newcomers to the northern Stafford area. At first glance, they may see its many stores, wide roads, and convenient subdivisions. That's modern Stafford, bedroom community to D.C. and Quantico Marine Corps Base. But Stafford County has a significant place in history, too.
Well-known local historian Jerrilynn Eby's Land of Herrings and Persimmons is a tremendous volume that chronicles the county's farming and industrial past, place by place, including Stafford County communities that were enveloped and lost when Quantico was established.
"I was born on the banks of the Rappahannock River. Taken Home to White Oak where I was raised and educated in the World's finest three-room university, White Oak School--now known as tribal member, artisan and historian D.P. Newton's Civil War Museum. Spent my time there with the other Patawomecks during World War Two getting lessons between the sounds of the big guns being tested at Dahlgren. They rattled the windows as the concussion came up through our Land. It was the sound of Freedom fighting back. We loved it. Attended Falmouth High and graduated from Stafford High. Graduated from a little Indian School in a place once known as Middle Plantation. Turned 78 nearly a year ago. Not much else to say, except, I am known as Johnny Mac."
The simple house of worship on White Oak Road, across from the White Oak Civil War Museum, has its historic roots in the separation of church and state and was a hub of Union Army activity in the winter of 1862-63.


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Air Products and Chemicals
Management Information Systems Facility | Trexlertown, PA
|For Air Products' Management Information Services (MIS) expansion, Bracy Contracting converted an existing office building into a printing facility with data entry and computer areas. Designed in-house by Air Products' staff, the project demanded the continuation of on-going operations critical to Air Products' business while construction took place.
|By building temporary walls to separate construction from on-going operations, working off-hours and weekends, maintaining strict dust and noise control and implementing personnel separation procedures for a safe work environment, Bracy kept the project moving smoothly.


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Views: 383, Replies: 7 » Jump to the end
Apr 30, 2016 4:20 AM CST
Apr 30, 2016 8:37 PM CST
Plants Admin
|Hi Charlotte, did you mean to add the above photo to the database entry? You enlarged your first photo and started a thread for it by adding the second image. The thread is now part of the Photos Forum.
[Last edited by Calif_Sue - Apr 30, 2016 8:38 PM (+)]
| Quote | Post #1133912 (2)
Apr 30, 2016 8:53 PM CST
|Oh, Suzanne. I *thought* I was adding it to the database. Am now thinking I will probably not attempt to do that any more as I'm a bit overwhelmed by this. Some people 'get' confused; I seem to 'stay' confused - especially when it comes to computers. Just was notified earlier today that another photo I submitted was incorrect (Huh in sandbox) - so .... am feeling like I'm not very good at the plant database thing.
Believe I'll stick to my butterfly photos from now on. That seems safer.
Thanks for pointing out the error. I apologize for the mix-up.
Apr 30, 2016 9:21 PM CST
Plants Admin
|It's not a problem, really! Sooo many people make the exact same mistake. Please don't stop adding photos, we love photos here!!
Just make sure when you are on the database page, use the Adding Actions section on the left side where you see the following:
» Upload a photo
» Upload a URL photo
» Post a comment
» Add an event report
» File a local report
When you enlarge one of the database images and scroll down and you'll see a subject line and message box just like you see when posting a comment to a thread. Above that box is says "Post a comment about this photo". I think many people just don't see that, all they see is the 'Upload an image' button.
May 1, 2016 6:34 PM CST
|Oh no don't stop please....It is a bit overwhelming but there are great people here to help if they can and if they can't there are more great people that can. There is that photo of yours in the sandbox that can be added easily. Click on it and it enlarges and just below it in blue letters...( I just had to go look at how to do it)... it says "Associate with a plant in the database" click on that and it goes through the steps. In this case I thought it might be Plantago erecta. Just type that into the box and it will come up to select it. Proceed to step two and presto it's into the correct plant in the DB. If your still not sure of the ID it can be posted to the ID forum, but I'm not sure how to do that, and someone will confirm or most likely find the proper ID. Your photos have been wonderful. Love all the Texas natives. People just don't take notice of lots of these let alone take a photo. The butterflies and cats are great too. I like all photos with the pollinators and other critters in them. My favorite photo of yours is this one .
Hamilton Square Garden, Historic City Cemetery, Sacramento California.
[Last edited by HamiltonSquare - May 1, 2016 6:39 PM (+)]
| Quote | Post #1135017 (5)
May 1, 2016 6:58 PM CST
|Thanks to all of you for your replies and words of encouragement @Calif_Sue. I will confess to having a bit of a "confidence crisis" yesterday. This is the first time I've ever shared my photos on any internet site, and it truly is 'a bit overwhelming' as you said @Leslieray. I'm not good at computers. (Old dog / new tricks syndrome). I just felt like I was making too many mistakes, and started to seriously wonder if this was going to work. But I've encountered a lot of nice people here, and I'm learning a lot. So no, after much consideration I've decided to keep trying. Please be patient and, no doubt I'll probably goof up again. But I'm going to keep at it.
May 1, 2016 7:09 PM CST
|Have successfully added the wayward photos to the Plant Database.
May 1, 2016 10:55 PM CST
Plants Admin
|« Garden.org Homepage
« Back to the top
« Forums List
« Photo of Drummond's Aster (Symphyotrichum drummondii var. texanum)
« Plant Photos Forum


text: being in perfect tune with your dog ~ calgary people & pet portrait photography Wednesday, January 30, 2013 it's always interesting when people & their pets are perfectly tuned in to each other . . . we love natural moments like these 🙂 « what do you do when the weather forecast looks like this? ~ the frozen Calgary pet photographerdiscovering the joy of walking your dog ~ day #31 complete ~ calgary pet photography »


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Good Vibes: Prosad brings the world back home
How does it feel to play in a stadium in front of 50,000 people?
Amazing. Yet eerily familiar, says Saskatoon musician, Prosad.
"It felt like I always imagined it would, actually. And it also felt like, this is what I'm supposed to be doing. I want to do this all the time."
The show was at the Dhaka International Folk Festival in Bangladesh. Prosad, who plays sitar, guitar and a host of other instruments, got the gig through a friend of a friend. He didn't really know what he was getting into, whether he'd be playing in front of a few people or a few thousand.
Artistically, he took a bit of a chance and performed his east-west fusion music, not knowing how it would go over.
"They like their traditional Bengali folk music and I thought maybe they'd like me to play something more traditional but they actually loved the fusion."
People who saw the show were humming his melodies in the hotel next day, Prosad says.
"Everyone there really loves music. There's such a passion for art and poetry and music.
Everybody sings, everybody has poetry memorized. It's a very romantic and artistic place."
On the same trip, he made contact with the band Chirkutt, who are like rock stars in Bangladesh.
"They kind of adopted me into their family," says Prosad. He ended up recording at their famous studio, coming away with seven tracks which will become his ninth original album, hopefully out in the fall.
"The producer wanted to break out and do something western and I already had this kind of draw toward the eastern so we're meeting toward the middle."
"That was an amazing adventure," Prosad says of his time in Bangladesh. He's had a few since graduating from Bishop James Mahoney High School, adopting the name Prosad which is Sanskrit for 'offering' and going down the road less travelled by playing sitar and gravitating toward India where he often travels.
He's studied sitar for 16 years but it's only recently that he's felt that his style, which includes rock, reggae and blues, is coming together.
"I'm really excited about it. It's going to be the bridge between the east and the west. Guitar and singer-songwriter and the sitar and kind of a mystical vibe."
In concert, Prosad picks up a wide range of instruments, building a sound with recorded loops and playing to the tracks.
A 90-minute set on the free stage at the jazz festival will showcase his unique style. Prosad will play digeradoo, saxophone and Indian flute in addition to electric guitar and electric sitar.
He's blending "all of those styles into something quite organic with a bit of a spiritual vibration from the sitar but also a grounded vibration just from growing up in Saskatchewan writing songs."
Looking ahead to the hometown show, Prosad remembers a line from T.S. Eliot:
"We shall not cease from exploration. And the end of all our exploring will be to arrive where we started and know the place for the first time."
He says he did feel out of place here learning the sitar and going on a spiritual path.
"But now I think the world has really opened up to yoga and spirituality and all these different things. So now, people are really embracing it and I do feel a sense of a homecoming."
Prosad also performs at the Saskatoon Reggae and World Music Fest, July 8 at W.E. Graham Park.
More on Prosad at www.prosadmusic.com


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An innovative mixtape featuring an epic intro, an adrenaline infused speed mix section, 3 JamJam remixes, 2 joints which JamJam featured on with an exclusive listen to JamJam's next feature Komalobe.
This mixtape is predominantly Naija Jamz with joints from South Africa, Ghana, Zimbabwe and Angola. Hear all the club bangers aswell as the upcomers making buzz on the streets of lagos!
►Single File MP3:
►Tracked Version:
34 DJ JamJam ft Artquake, Rugged Man, Klever Jay, K-Solo – Luv Naija Music (Komalobee)


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Information For
Housing Services > Assignments > Midyear Housing
If you're looking to live on campus, no date is too late. Housing Services assigns students to campus housing year-round on both the Lincoln Park and Loop campuses. Please Note: Housing is available for fall, winter and spring quarters! Learn more about midyear housing in this brochure. If you are interested in applying or have questions, please call our office at (773) 325-7196.
Applying to campus housing is easy. Just fill out the housing agreement at myhousing.depaul.edu.
A $400 prepayment must be made in order for your agreement to be complete. If you are under 18 years old at the time of applying, you will need to fill out the parent/guardian voucher and return the form to our office by sending it via postal mail or by scanning and emailing it.
Be sure to read and understand the 2018-19 Terms and Conditions.
Centennial Hall, Suite 301
2345 North Sheffield Avenue
Chicago, IL 60614
(773) 325-7196
Monday - Friday 9 a.m. - 5 p.m.
2001-2012 DePaul University | Disclaimer | Contact Us | Emergency Plan | Consumer Information | Privacy Statement Editor Login


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Gap moves to Church Street
Scheduled to open this fall
Gap moves to Church Street
Scheduled to open this fall
The retail store Gap is moving from it's current location in the Burlington Town Center Mall to 1 Church Street in Burlington.
The new 10,000 square foot space is where Ann Taylor was formerly located. Since 2008 when Ann Taylor moved out the space has been empty.
The second floor space will be the new home of Gap, Baby Gap, and Athleta wear in Burlington.
Church Street Marketplace officials say that the addition of Gap brings the marketplace to 99% occupancy.
The new location is scheduled to open this fall.


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Invasion privacy: penalties remedies, Invasion privacy: penalties remedies canada zealand tort cases privacy ingredient action.


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Following is a speech by the Secretary for Financial Services and the Treasury, Mr Frederick Ma, at the Annual Dinner of The Hong Kong Institute of Chartered Secretaries (HKICS) today (January 17):
New Year Wishes - Corporate Governance in Hong Kong
Richard, Distinguished Guests, Ladies and Gentlemen,
Good evening. Let me, first of all, wish you a happy and prosperous 2006!
I am delighted to be here tonight to address the Hong Kong Institute of Chartered Secretaries Annual Dinner 2006. This is indeed a great opportunity for me to share with a group of key professionals in Hong Kong - company secretaries, my thoughts in the area of corporate governance (CG).
Importance of Corporate Governance
CG is close to the heart of anyone in our business community, particularly all of you sitting here who have been taking good care of the CG system in your companies. Since the collapse of Enron and Worldcom, perhaps no one in the business sector can afford not to take CG seriously. While good CG may not on its own ensure the prosperity of companies, poor CG can definitely put companies in jeopardy. The Chief Executive mentioned in his maiden Policy Address last October that -
"Hong Kong is a leading international financial centre in the Asia-Pacific region. We will continue to consolidate this position, which is of vital importance to our prosperity. ...... The Government upgrades the quality of financial markets by enhancing the regulatory regime and the promotion of good corporate governance. ......".
These succinct remarks have clearly demonstrated the importance the Government has attached to the subject of CG. As we all know, the "formula" is as simple as that - Good CG enables Hong Kong to stay ahead as a preferred choice for quality issuers to raise capital, and for quality investors, both local and overseas, to invest their money, thereby strengthening Hong Kong's position as an international financial centre. The community as a whole, from individual companies and shareholders to the general public, will benefit one way or another. Some figures on our financial markets will give you a feel of how the formula is working -
* As at end-December 2005, Hong Kong's stock market ranked eighth in the world and second in Asia in terms of market capitalisation.
* In terms of equity raised in the first 11 months of 2005, Hong Kong ranked fourth globally and first in Asia, ahead of Tokyo.
* Hong Kong is the premier international financial centre for the Mainland. The vast majority of Mainland enterprises listed outside the Mainland are listed on our stock exchange. Over past years, Mainland enterprises have raised more than HK$1,080 billion in Hong Kong.
* In terms of asset management, Hong Kong is rising fast as Asia's leading asset management centre. In 2004, our total asset management business amounted to HK$ 3,618 billion.
For 2006, I have a few wishes relating to CG and would like to share them with you.
First Wish: Government and Regulators
Let me start with my first CG wish, which is made for the Government and regulators. Our general philosophy has always been that: both Government and regulators should strike the right balance between effective regulation and market developments, thereby making our financial market not only fair and orderly, but also a vibrant and growing one. With this in mind, I wish that the Government and the regulators will continue to -
(a) put in place a regulatory regime, which is on a par with the evolving international standards;
(b) ensure that effective sanctions and remedies are available if and when things go wrong; and
(c) provide an environment that encourages and facilitates the business sector to pursue good CG.
Over the past few years, there has been much progress in these areas. Just to quote some examples -
(a) The Securities and Futures Ordinance
Following the roll-out of the Securities and Futures Ordinance (SFO) in April, 2003, our regulatory regime for the securities and futures sector has been overhauled and modernised, and is now in line with the international practices. For instance, we have criminalised filing or disclosure of false, or misleading, listing documents, given that proper disclosure of information by listed companies is essential to orderly market transactions and protection of investors. The Securities and Futures Commission is also given enhanced powers to combat corporate misconduct.
(b) Code on Corporate Governance Practices
The Code on Corporate Governance Practices launched by HKEx, which has been benchmarked against the best prevailing market practices and international standards, came into effect in January, 2005. The Code contains two levels of recommendations -
* Code Provisions: A listed issuer is required to comply or explain its non-compliance in its Corporate Governance Report. Examples include holding of board meetings at least four times a year at approximately quarterly intervals, and the establishment of a remuneration committee with a majority of its members being independent non-executive directors (INEDs); and
* Recommended Best Practices: Compliance is encouraged but disclosure of non-compliance is voluntary. Examples include having at least one-third of the board members being INEDs.
By adopting a comply-or-explain rather than a prescriptive approach, the Code can encourage corporate officers like you to design the best CG measures that suit a company's specific circumstances.
In the coming year, we will continue with our work to spearhead an important legislative exercise, namely to give statutory backing to major listing requirements such as disclosure of price-sensitive information and financial reporting and other periodic disclosure. The proposals aim to further enhance the standard of corporate disclosure. Our plan is to introduce the legislative proposals into the Legislative Council (Legco) within this year.
More importantly, later this year, we will launch the planned rewrite of the Companies Ordinance, a law book which many of you know is several inches thick. Other than modernising the relevant provisions so as to help reduce business costs, such as facilitating companies to introduce electronic communications, the rewrite has another major objective. That is to implement the remaining CG-related recommendations made by the Standing Committee on Company Law Reform, such as changes to the procedures in the nomination and election of directors. We believe that with the implementation of these recommendations, which are important building-blocks of a stronger CG regime, Hong Kong's position as an international business and financial centre will be further enhanced. We will closely involve the stakeholders, including the professional bodies like the HKICS, throughout the rewrite. Your participation and advice will definitely help us to ensure that the new Companies Ordinance would be a quality piece of work that meets Hong Kong's circumstances.
Second Wish - Shareholders
My second wish goes to shareholders. Shareholders own companies, and have the legitimate rights to say how the companies should operate. Since their own investments are involved, it is natural to expect them to have a watchful eye on the CG of their companies. The key is to properly equip them and promote awareness. Shareholders should be encouraged to monitor the operations of a company. How to read the financial statements and annual reports? What is the environment in which the company is operating? What are the internal checks and balances, and any areas for improvement? Shareholders should also understand their rights, including their rights to attend, speak and vote at general meetings. Numerous publicity campaigns and public education are organised by the regulators on an on-going basis, with a view to helping shareholders to be more proactive in monitoring their companies and exercising their rights in a positive manner.
The equipping, or empowerment, of shareholders so that they will be able to exercise their rights is also an important element of a good CG system. In Hong Kong, Schedule 3 of the Companies (Amendment) Ordinance 2004 has become operative in July, 2005. The new legislation provides an avenue for shareholders to seek statutory derivative action on behalf of a company on any expropriation or other wrongs done. The court is also now empowered to award damages to shareholders whose interests have been unfairly prejudiced.
Third Wish - Directors
My third CG wish is made for directors. Good CG at the company level can hardly be achieved by a robust regulatory framework and vigilant shareholders alone. Good CG has therefore to start from the company boardroom.
Directors are responsible for overseeing the affairs and activities of a company. Representing shareholders of a company, they are entrusted with the responsibility of running the corporation, including acting as a check on corporate management. To champion CG at the company level, it is of utmost importance to, first of all, help directors understand their roles, duties and obligations. There are already many useful and handy references on these duties around us, such as the Guidelines of Directors' Duties drawn up by the Standing Committee on Company Law Reform. What follows is appropriate training both on first appointment and a continuous basis for directors to help them acquire a thorough understanding of the prevailing laws and rules in the market as well as their obligations and rights. The Code on Corporate Governance Practices, which I have mentioned earlier, has included a code provision for comprehensive and formal induction for all newly appointed directors. There is also a recommended best practice on continuous training for all directors.
Fourth Wish: The Professionals
Now let me turn to the fourth and also the last CG wish, which is made for the professionals. For example, accountants perform a variety of roles, as auditors who conduct an external, independent review of the financial reporting process and company financial statements, and as professional accountants in business, who may act as finance controllers, internal auditors or compliance officers. Irrespective of the exact roles they undertake, they are all important gatekeepers. Shareholders, creditors, investors and even the regulators rely on their report to ensure the integrity of the financial statements of a company. I trust that the profession will continue to meet or even exceed stakeholders' expectations, and safeguard the "purity" of the information in company accounts.
The Government will also continue to ensure that the accounting profession is subject to an effective, transparent and independent regulatory regime. In this regard, together with the Hong Kong Institute of Certified Public Accountants, we have done a lot in the past few years, such as the enactment of the Professional Accountants (Amendment) Ordinance 2004 which opens up the governance body, as well as the investigatory and disciplinary systems, of the Institute. Another initiative being taken forward is the proposal to establish a statutory Financial Reporting Council, which will be tasked to carry out independent investigation into any irregularities concerning the auditors of listed corporations and to conduct enquiry into the financial reports of listed corporations to ensure their compliance with relevant regulatory requirements.
Of course, I cannot complete the CG picture if I miss the chartered, or company, secretaries, who also play an important role. The secretary of a company has to undertake a wide range of duties, both statutory and non-statutory. These include completing and filing of various returns and documents, setting up a system for proper keeping and update of records, advising on relevant procedures such as holding of general meetings or board meetings, acting as the primary source of counsel to directors over company affairs and so on. Furthermore, the company secretary is the one who knows how the "nuts and bolts" of the companies' work day to day. Indeed, one may say that he or she is the de facto guardian of a company's CG through ensuring compliance with the relevant requirements and upholding business ethics. He or she is also the one who is expected to possess skills and expertise in areas such as company law, financial management, company secretaryship and administration.
Without the good work and active participation of a group of professional company secretaries like you, we would not be able to achieve a robust CG regime. I therefore wish that company secretaries will continue to push for good CG in handling company affairs. While not a fortune teller, I am very confident that this wish would materialise, to a large extent because of the outstanding job done by the HKICS. Over the years, the Institute has strived to enhance its members' as well as other stakeholders' understanding of the new CG requirements. For example, there is a biennial CG Conference to offer an opportunity for renowned local and overseas experts to share their insights on typical CG issues. In 2004, the HKICS also introduced an enhanced continuing professional development programme, endorsed by the HKEx, that is designed to equip its members working in listed companies with the current knowledge and skills to implement effectively the new CG regulations. The HKICS is also a strong promoter of good CG culture through various means such as publication of newsletters and guidelines, organisation of workshops and so on. The newly released second edition of the Report on "The Duties and Responsibilities of Independent Non-Executive Directors of Hong Kong Main Board Listed Companies" is another good example of HKICS's invaluable contribution to the promotion of good CG practices in Hong Kong.
Indeed, I am confident that all the CG wishes I mentioned earlier will come true in 2006. My confidence comes from the on-going and strong commitments made by all relevant parties, including regulators, accountants, shareholders, directors and company secretaries, to further upgrade Hong Kong's CG regime and provide an environment for it to grow and prosper. Let us all continue to work together towards this objective in 2006. Thank you very much.
Ends/Tuesday, January 17, 2006
Issued at HKT 20:01


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The explosive account of how Republican legislators and political operatives fundamentally rigged American democracy through redistricting.
With Barack Obama's historic election in 2008, pundits proclaimed the Republicans as dead as the Whigs of yesteryear. Yet even as Democrats swooned, a small cadre of Republican operatives began plotting their comeback with a simple yet ingenious plan. These men had devised a way to take a tradition of dirty tricks—known to political insiders as "ratf**king"—to an unprecedented level. Flooding state races with a gold rush of dark money, the Republicans reshaped state legislatures where the power to redistrict is held. Reconstructing this previously untold story, David Daley examines the far-reaching effects of this programme, which has radically altered America's electoral map and created a firewall in the House. Ratf**ked pulls back the curtain on one of the greatest heists in American political history.


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Construction firm Costain has made an increased all-share offer approach to struggling business services group Mouchel, valuing the company at about £150m.
Costain said it would offer 0.5947 new shares for each Mouchel share. The offer values each Mouchel share at about 135 pence per share, based on Wednesday's closing prices.
In December, Mouchel rejected an approach from Costain comprising 0.5135 new Costain shares for each Mouchel share.
Costain said on Thursday it was in the interests of both sets of shareholders for the board of Mouchel to enter into discussions with Costain as soon as possible.
City A.M. Reporter


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AIFS Study Abroad in Paris, ILCF: Semester or Academic Year
Video and Photos
Earn up to 17 credits studying in the beautiful city of Paris. Study French Language at one of four levels and take additional courses in French Culture and Civilization taught in English or French.
Live in a homestay, apartment or student residence, all with a meal plan and internet access. Enjoy cultural and social activities such as guided city walks, visits to the many museums and cultural sites of Paris, French cooking classes, opera and ballet performances, and day trips to Monet's Garden in Giverny and the famous Château de Versailles. Optional excursions include a 2-day trip to Normandy and Brittany, and a 2-day trip to the Loire Valley.
AIFS students have the ability to take art and music courses through the Académie de Port Royal and Schola Cantorum respectively. Volunteer opportunities are also available.
- Monet's Gardens in Giverny
- Half day trip to Château de Versailles
- Visits to local Museums
- Walking tour of the Montmartre district


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What are friends for? Sping Cleaning/Protecion


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