Optical forward-scattering for detection of Listeria monocytogenes and other Listeria species

We demonstrate here the development of a non-invasive optical forward-scattering system, called 'scatterometer' for rapid identification of bacterial colonies. The system is based on the concept that variations in refractive indices and size, relative to the arrangement of cells in bacterial colonies growing on a semi-solid agar surface will generate different forward-scattering patterns. A 1.2-1.5mm colony size for a 1mm laser beam and brain heart infusion agar as substrate were used as fixed variables. The current study is focused on exploring identification of Listeria monocytogenes and other Listeria species exploiting the known differences in their phenotypic characters. Using diffraction theory, we could model the scattering patterns and explain the appearance of radial spokes and the rings seen in the scattering images of L. monocytogenes. Further, we have also demonstrated development of a suitable software for the extraction of the features (scalar values) calculated from images of the scattering patterns using Zernike moment invariants and principal component analysis and were grouped using K-means clustering. We achieved 91-100% accuracy in detecting different species. It was also observed that substrate variations affect the scattering patterns of Listeria. Finally, a database was constructed based on the scattering patterns from 108 different strains belonging to six species of Listeria. The overall system proved to be simple, non-invasive and virtually reagent-less and has the potential for automated user-friendly application for detection and differentiation of L. monocytogenes and other Listeria species colonies grown on agar plates within 5-10 min analysis time.     

Fine Structure of Elytral Punctures and Diffraction Gratings in Korean Sericinae (Coleoptera, Melolonthidae)

ABSTRACT The surface structures of the elytra in twenty two species belonging to the subfamily Sericinae (Coleoptera, Melolonthidae) were observed by scanning electron microscopy. For viewing specific characters, elytral punctures and diffraction gratings on interstitial surface were focused on. The elytral punctures of observed Sericid beetles have three representative forms: horseshoe, ocellus, and crease types. The diffraction gratings of the elytra show three distinctive patterns: regular and parallel, discontinued, and interconnected and curved. These patterns of elytral punctures and diffraction gratings considered to be useful for identification.     

Discrimination between Acartia (Copepoda: Calanoida) species using their diffraction pattern in a position, rotation invariant digital correlation

Digital images of Acartia discaudata, A. clausi, A. margalefiand A. tonsa were processed to obtain their diffraction pattern.To discriminate between species and sex all diffraction patternswere correlated with a spatial filter, invariant to positionand rotation, for each Acartia male and female. This filterwas made up using a combination of different images of eachspecies and sex. Considering the great similarity between thecopepod species used in this work and between the male and femaleof each species the results obtained are very good. It is concludedthat the method used to discriminate between species of thiscongeneric group can be very useful for the development of anautomated system for the identification of copepods.     

Low dose electron microscopy of the crotoxin complex thin crystal

The crotoxin complex from Crotalus d. terrificus rattlesnake venom was crystallized in the form of thin platelets. These crystals were prepared by the glucose embedding technique and examined by low dose electron microscopy. Electron diffraction patterns and images have been recorded to 2.2 and 4.5 A, respectively. By a combination of electron and X-ray diffraction techniques, the space group of this crystal was determined to be P4(2)22 with eight crotoxin complex molecules in one unit cell with dimensions of 38.8 A x 38.8 A x 256.8 A. The Patterson maps and the symmetry reliability factors calculated from the electron diffraction intensities clearly showed the existence of three types of electron diffraction patterns in different crystals. The phases in the computer-calculated transform of the low dose images also show the variation in symmetry among crystals. These phenomena are explained by the presence of crystals consisting of one-half, three-quarter and one unit cell in thickness. The interpretation of the computer reconstructed two-dimensional density map was limited, partly because of the similarity in density between the protein and the embedding glucose and partly because of the non-uniqueness in relating projected structure to the three-dimensional structure.     

Label-free identification of bacterial microcolonies via elastic scattering

Label‐free microcolony identification via elastic light scattering was investigated for three different genera: Salmonella enterica serovar Montevideo, Listeria monocytogenes F4244, and Escherichia coli DH5α. Microcolonies were defined as bacterial colonies in their late‐lag phase to early‐exponential phase with the diameter range of 100–200 µm. To link biophysical characteristics with corresponding scattering patterns, a phase contrast microscope and a confocal displacement meter were used to measure the colony diameter and its 3D height profile. The results indicated that the growth characteristics of microcolonies were encoded in their morphologies which correlated to the characteristic diffraction patterns. Proposed methodology was able to classify three genera based on comprehensive phenotypic map which incorporated growth speed, ring count, and colony diameter. While the proposed method illustrated the possibility of discriminating microcolonies in their early growth stage, more thorough biophysical understanding is needed to expand the technology to other species. Biotechnol. Bioeng. 2011; 108:637–644. © 2010 Wiley Periodicals, Inc.     

The crystalline glycoprotein cell wall of the green alga Chlorogonium elongatum: a structural analysis.

Members of the Chlamydomonaceae, mostly single-celled green algae, have been shown to contain a crystalline glycoprotein cell wall component. Most of the species examined fall into a class of algae whose walls have an identical crystalline unit cell. Chlorogonium elongatum has been chosen as a representative of this class in order to investigate in more detail its cell wall structure. The alga has a spindleshaped cell wall which retains its asymmetric shape on isolation. Sections from walls fized in the presence of tannic acid clearly reveal a regular subunit monolayer, about 20 nm thick, within the wall. Sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis shows the presence of at least 2 major glycoprotein species in the wall. Negatively stained purified cell walls demonstrate the crystalline nature of the cell wall. Optical diffraction of bright-field images and direct electron diffraction both give clear diffraction patterns whose spacings extend out to 3 nm and fall on a reciprocal lattice whose vectors describe a 2-dimensional unit cell within the wall 21.5 nm X 7.0 nm and an included angle of 80 degrees. Lattice defects within the cell wall are revealed by both negative staining and surface replication. Through-focal series were used to choose images with the optimal degree of underfocus for image processing. Linear integration and optical filtering of such images gave essentially the same result. A similar image was also obtained by computing the autocorrelation function of the amplitudes in the electron-diffraction pattern and the optical-diffraction pattern of the in-focus image. On the basis of these data a 2-dimensional model of the crystalline cell wall layer is presented.     

Applying machine learning based on multiscale classifiers to detect remote phenology patterns in Cerrado savanna trees

Plant phenology is one of the most reliable indicators of species responses to global climate change, motivating the development of new technologies for phenological monitoring. Digital cameras or near remote systems have been efficiently applied as multi-channel imaging sensors, where leaf color information is extracted from the RGB (Red, Green, and Blue) color channels, and the changes in green levels are used to infer leafing patterns of plant species. In this scenario, texture information is a great ally for image analysis that has been little used in phenology studies. We monitored leaf-changing patterns of Cerrado savanna vegetation by taking daily digital images. We extract RGB channels from the digital images and correlate them with phenological changes. Additionally, we benefit from the inclusion of textural metrics for quantifying spatial heterogeneity. Our first goals are: (1) to test if color change information is able to characterize the phenological pattern of a group of species; (2) to test if the temporal variation in image texture is useful to distinguish plant species; and (3) to test if individuals from the same species may be automatically identified using digital images. In this paper, we present a machine learning approach based on multiscale classifiers to detect phenological patterns in the digital images. Our results indicate that: (1) extreme hours (morning and afternoon) are the best for identifying plant species; (2) different plant species present a different behavior with respect to the color change information; and (3) texture variation along temporal images is promising information for capturing phenological patterns. Based on those results, we suggest that individuals from the same species and functional group might be identified using digital images, and introduce a new tool to help phenology experts in the identification of new individuals from the same species in the image and their location on the ground.     

Frog skeletal muscle thick filaments are three-stranded

A procedure has been developed for isolating and negatively staining vertebrate skeletal muscle thick filaments that preserves the arrangement of the myosin crossbridges. Electron micrographs of these filaments showed a clear periodicity associated with crossbridges with an axial repeat of 42.9 nm. Optical diffraction patterns of these images showed clear layer lines and were qualitatively similar to published x-ray diffraction patterns, except that the 1/14.3-nm meridional reflection was somewhat weaker. Computer image analysis of negatively stained images of these filaments has enabled the number of strands to be established unequivocally. Both reconstructed images from layer line data and analysis of the phases of the inner maxima of the first layer line are consistent only with a three-stranded structure and cannot be reconciled with either two- or four-stranded models.     

Electron microscopic and optical diffraction analysis of the structure of scorpion muscle thick filaments

We rapidly and gently isolated thick filaments from scorpion tail muscle by a modification of the technique previously described for isolating Limulus thick filaments. Images of negatively stained filaments appeared to be highly periodic, with a well-preserved myosin cross-bridge array. Optical diffraction patterns of the electron micrograph images were detailed and similar to optical diffraction patterns from Limulus and tarantula thick filaments. Analysis of the optical diffraction patterns and computed Fourier transforms, together with the appearance of the filaments in the micrographs, suggested a model for the filaments in which the myosin cross-bridges were arranged on four helical strands with 12 cross-bridges per turn of each strand, thus giving the observed repeat every third cross-bridge level. Comparison of the scorpion thick filaments with those isolated from the closely related chelicerate arthropods, Limulus and tarantula, revealed that they were remarkably similar in appearance and helical symmetry but different in diameter.     

Densely packed beta-structure at the protein-lipid interface of porin is revealed by high-resolution cryo-electron microscopy

Porin is an integral membrane protein that forms channels across the outer membrane of Escherichia coli. Electron microscopic studies of negatively stained two-dimensional porin crystals have shown three stain accumulations per porin trimer, revealing the locations of pores spanning the membrane. In this study, reconstituted porin lattices embedded in glucose were investigated using the low-dose technique on a cryo-electron microscope equipped with a helium-cooled superconducting objective lens. The specimen temperature was maintained at 5 K to yield an improved microscopic and specimen stability. Under these conditions, we obtained for the first time electron diffraction patterns from porin lattices to a resolution of 3.2 A and images showing optical diffraction up to a resolution of 4.9 A. Applying correlation averaging techniques to the digitized micrographs, we were able to reconstruct projected images of the porin trimer to a resolution of up to 3.5 A. In the final projection maps, amplitudes from electron diffraction and phases from these images were combined. The predominant feature is a high-density narrow band (about 6 A in thickness) that delineates the outer perimeter of the trimer. Since the molecule consists of almost exclusively beta-sheet structure, as revealed by spectroscopic data, we conclude that this band is a cylindrical beta-pleated sheet crossing the membrane nearly perpendicularly to its plane. Another intriguing finding is a low-density area (about 70 A2) situated in the centre of the trimer.     

Using phenological cameras to track the green up in a cerrado savanna and its on-the-ground validation

Plant phenology has gained new importance in the context of global change research, stimulating the development of novel technologies for phenological observations. Regular digital cameras have been effectively used as three-channel imaging sensors, providing measures of leaf color change or phenological shifts in plants. We monitored a species rich Brazilian cerrado savanna to assess the reliability of digital images to detect leaf-changing patterns. Analysis was conducted by extracting color information from selected parts of the image named regions of interest (ROIs). We aimed to answer the following questions: (i) Do digital cameras capture leaf changes in cerrado savanna vegetation? (ii) Can we detect differences in phenological changes among species crowns and the cerrado community? (iii) Is the greening pattern detected for each species by digital camera validated by our on-the-ground leafing phenology (direct observation of tree leaf changes)? We analyzed daily sequences of five images per hour, taken from 6:00 to 18:00 h, recorded during the cerrado main leaf flushing season. We defined 24 ROIs in the original digital image, including total or partial regions and crowns of six plant species. Our results indicated that: (i) for the studied period, single plant species ROIs were more sensitive to changes in relative green values than the community ROIs, (ii) three leaf strategies could be depicted from the species' ROI patterns of green color change, and (iii) the greening patterns and leaf functional groups were validated by our on-the-ground phenology. We concluded that digital cameras are reliable tools to monitor high diverse tropical seasonal vegetation and it is sensitive to inter-species differences of leafing patterns.     

Alignment and merging of electron microscope images of frozen hydrated crystals of the T4 DNA helix destabilizing protein gp32*I.

Low dose cryoelectron microscopy has been used to record images and electron diffraction patterns of frozen hydrated crystals of the single-stranded DNA binding protein gp32*I. Fourier transforms from 13 image areas, corresponding to approximately 40,000 unit cells, were aligned by a minimal phase residual search and merged by vector addition in reciprocal space. Phases from the resulting composite transform were combined with amplitudes from electron diffraction patterns to reconstruct the projected mass density of the gp32*I crystal at 8.4 A resolution.     

Photoluminescence emission from nanostructured porous preparations of CdS–ZnTiO3 assembled nanoparticles

This study reports the structural and optical properties of CdS/ZnTiO₃ nanocomposites prepared using a chemical bath and different titanate concentrations. Commercial ZnTiO₃ nanoparticles were introduced into a chemical bath that had been used to produce CdS semiconductor nanoparticles (NPs). Here, the growing CdS crystallites precipitated onto the suspended zinc titanate NPs. X‐ray diffraction patterns revealed that samples of CdS/ZnTiO₃ nanopowders were made of cubic ZnTiO₃ and hexagonal CdS wurtzite. The morphology of the particles was studied using transmission electron microscopy and scanning electron microscopy images. These images demonstrated the different characteristics of the CdS/ZnTiO₃ nanocomposites and their dependence on titanate concentration when placed into the CdS‐growing solution. Photoluminescence spectra showed three main emission bands for the electron transitions in the CdS/ZnTiO₃ composite. This composite produced three photoluminescence bands, the intensities of which depended on composite shape, which in turn depended on the relative concentrations of CdS and ZnTiO₃.     

Live biospeckle laser imaging of root tissues

Live imaging is now a central component for the study of plant developmental processes. Currently, most techniques are extremely constraining: they rely on the marking of specific cellular structures which generally apply to model species because they require genetic transformations. The biospeckle laser (BSL) system was evaluated as an instrument to measure biological activity in plant tissues. The system allows collecting biospeckle patterns from roots which are grown in gels. Laser illumination has been optimized to obtain the images without undesirable specular reflections from the glass tube. Data on two different plant species were obtained and the ability of three different methods to analyze the biospeckle patterns are presented. The results showed that the biospeckle could provide quantitative indicators of the molecular activity from roots which are grown in gel substrate in tissue culture. We also presented a particular experimental configuration and the optimal approach to analyze the images. This may serve as a basis to further works on live BSL in order to study root development.     

Scale‐dependent thermal tolerance variation in Australian mountain grasshoppers

Physiological variation among and within species is thought to play a key role in determining distribution patterns across environmental gradients. We tested inter‐ and intraspecific variation in cold and heat tolerances for three grasshopper species (genus Kosciuscola) with overlapping elevation distributions, across their respective ranges in the Australian mountains. Of the three cold tolerance traits measured, the critical thermal minimum was the only trait to vary among species, with greater cold tolerance associated with a distribution extending to a higher elevation. Cold tolerance limits were regularly exceeded in exposed microhabitats, suggesting a role for cold adaptation in structuring species distribution patterns. In contrast to cold tolerance, heat tolerance variation was primarily partitioned within species. For two species, populations from treeless alpine habitat were more heat tolerant than their lower‐elevation counterparts, supporting recent models that suggest greater exposure to temperature extremes at higher elevations. These contrasting patterns of physiological variation among and within species emphasise the importance of considering variation within species when attempting to understand how species distributions are affected by thermal extremes.     

Structure of CaATPase: electron microscopy of frozen-hydrated crystals at 6 A resolution in projection

Thin, three-dimensional crystals of CaATPase have been studied at high resolution by electron crystallography. These crystals were grown by adding purified CaATPase to appropriate concentrations of lipid, detergent and calcium. A thin film of crystals was then rapidly frozen and maintained in the frozen-hydrated state during electron microscopy. The resulting electron diffraction patterns extend to 4.1 A resolution and images contain phase data to 6 A resolution. By combining Fourier amplitudes from electron diffraction patterns with phases from images, a density map has been calculated in projection. Comparison of this map from unstained crystals with a previously determined map from negatively stained crystals reveals distinct contributions from intramembranous and extramembranous protein domains. On the basis of this distinction and of the packing of molecules in the crystal, we have proposed a specific arrangement for the ten alpha-helices that have been suggested as spanning the bilayer.     

Two-dimensional structure of plant light-harvesting complex at 3.7 A [corrected] resolution by electron crystallography

The structure of the light-harvesting chlorophyll a/b-protein complex has been determined at 3.7 A resolution in projection by electron diffraction, electron microscopy and image analysis. Diffraction patterns and high-resolution spotscan images of two-dimensional crystals stabilized with tannin were recorded at low temperature. Phases of structure factors were obtained directly by image processing, after correction of the images for lattice distortions, defocus and beam tilt. Amplitudes were measured by electron diffraction. The projection map shows the detailed structure of the trimeric complex, suggesting the positions of two domains of potential structural and functional homology, of one membrane-spanning alpha-helix approximately perpendicular to the membrane plane and of several tightly bound lipid molecules.     

ERRATUM

The legends to Figures 9, 10 and 11 were incorrect. They shouldhave read: Fig.9. Complete Linkage dendrograms with all the variables ofsegmentation for the organisms (b) and their diffraction patterns(a). Flg. 10. Complete linkage dendrograms without variables 4 and5 for the organisms (b) and their diffraction patterns (a). Fig. ll. Complete linkage dendrograms for the organisms witha major degree of segmentation (b) and their diffraction patterns(a).     

采用Strauss-Hardcore模型研究不同导管构型被子植物的导管空间分布特征

被子植物木质部导管的分布格局非常多样, 并且与木质部的输水功能有密切的联系, 然而在木材解剖学中对导管分布格局往往采用定性描述, 不利于分析该特征与物种的水力功能、生态地理分布的关系。该文采用点格局分析方法, 依据木材孔型、导管空间排列和导管群集度三类木材宏观结构特征组合, 选取不同导管分布类型的17种代表性阔叶树种, 利用Strauss-Hardcore模型对其木质部横切面解剖影像进行定量分析。Strauss-Hardcore模型能够很好地拟合木质部中导管二维空间位点的分布特征, 该模型的3个参数: 硬核距离、局部聚集距离、点对交互作用强度(局部聚集指数)都有着明确的生物学意义。传统解剖学对导管构型的定性分类同模型相比不能准确表现被子植物的木质部导管空间分布特征, Strauss-Hardcore模型的局部聚集度指数主要受导管群集度影响, 尤其是复导管和导管团的存在都会增大导管小尺度聚集程度。对散孔材、半环孔材的生长轮及环孔材的晚材部分解剖图像分析表明, 导管以单导管为主且没有明显分布方向的散孔材树种, 其木质部导管点对交互作用强度为负值, 局部聚集指数一般小于0.4, 导管空间分布依次在3个局部尺度表现出排斥-排斥-随机格局; 而导管具有径向、弦向、锯齿形等明显目视识别特征的物种, 无论孔型和是否以单复导管为主, 其导管点对交互作用强度为正值, 局部聚集指数均大于0.4, 导管依次在3个局部尺度上表现出排斥-聚集-随机的分布格局。采用点过程模型有利于准确描述导管二维空间分布规律, 增强对导管空间格局形成机理的理解, 可有力地支撑木质部三维导管系统的理论研究和木质部结构-功能的实验研究。     

Three-dimensional structure of the crystalloid in the microbody of Kloeckera sp.: composite crystal model.

Electron microscopic investigations using the cryosectioning technique, together with electron diffraction, optical diffraction, and computer simulation, were carried out for the determination of the intrinsic structure of the crystalloid in the microbody of Kloeckera sp. The lattice images seen in the cryosections could be changed from one to another by tilting the specimen at an appropriate angle, the images obtained being well consistent with those obtained by computer simulation. The electron diffraction patterns also agreed with those obtained by optical diffraction. The results demonstrated that the crystalloid was composed of two different types of particles, large and small ones, arranged alternately and making up the composite crystal of rock salt structure. Large particles seemed to consist of alcohol oxidase molecules, while the small particles were presumably made up of catalase molecules.