The Good,the Bad,and the Ugly: The Influence of Skull Reconstructions and Intraspecific Variability in Studies of Cranial Morphometrics in Theropods and Basal Saurischians

Several studies investigating macroevolutionary skull shape variation in fossil reptiles were published recently, often using skull reconstructions taken from the scientific literature. However, this approach could be potentially problematic, because skull reconstructions might differ notably due to incompleteness and/or deformation of the material. Furthermore, the influence of intraspecific variation has usually not been explored in these studies. Both points could influence the results of morphometric analyses by affecting the relative position of species to each other within the morphospace. The aim of the current study is to investigate the variation in morphometric data between skull reconstructions based on the same specimen, and to compare the results to shape variation occurring in skull reconstructions based on different specimens of the same species (intraspecific variation) and skulls of closely related species (intraspecific variation). Based on the current results, shape variation of different skull reconstructions based on the same specimen seems to have generally little influence on the results of a geometric morphometric analysis, although it cannot be excluded that some erroneous reconstructions of poorly preserved specimens might cause problems occasionally. In contrast, for different specimens of the same species the variation is generally higher than between different reconstructions based on the same specimen. For closely related species, at least with similar ecological preferences in respect to the dietary spectrum, the degree of interspecific variation can overlap with that of intraspecific variation, most probably due to similar biomechanical constraints.     

Regeneration of halved embryonic lower first mouse molars: correlation with the distribution pattern of non dividing IDE cells, the putative organizers of morphogenetic units, the cusps

Recently we demonstrated that non-cycling, cap-stage, mouse molar inner dental epithelial (IDE) cells corresponding to the primary enamel knot (EK) area underwent a coordinated temporo-spatial patterning leading to their patchy irregular segregation at the tips of the forming cusps. These non-cycling cells were suggested to perhaps represent the organizers of the morphogenetic units (OMU), the cusps. The present study has analyzed the regenerative capacity of halved cap-stage first lower mouse molars through three dimensional (3D) reconstructions. Partial regeneration of the anterior half and possible complete regeneration of the posterior half were documented. Using BrdU (5-bromo-2'-deoxyuridine) labeling and 3D reconstructions of the IDE, we have correlated the patterns of cusp regeneration with the distribution of BrdU negative IDE cells. These data support a morphogenetic role for the non-cycling IDE cells.     

Use of the AO plate for immediate mandibular reconstruction in cancer patients

Free vascularized bone grafts have revolutionized mandibular reconstruction, yet their use in all mandibulectomy patients is not always necessary. A recently developed alternative to bony reconstruction has been the use of the AO reconstruction plate. We compared the use of the AO reconstruction plate with immediate free bone graft mandibular reconstruction in 31 patients. Reconstruction plates were used in 20 and immediate free bone grafts were used in 11 patients. The overall success rate for use of the plate was 15 of 20 (75 percent). There were 6 anterior reconstructions, of which only 2 (33 percent) were successful. This is opposed to 13 of 14 (93 percent) lateral reconstructions that were successful in lateral plate placements. There were 11 immediate composite free flaps: 4 iliac crest, 4 scapula, 2 fibula, and 1 composite radial forearm flaps. Six repairs were for anterior defects, and there were 5 full-thickness defects, 3 of which were in the anterior position. All 11 flaps were successful. In conclusion, we believe the reconstruction plates are a useful adjunct for mandibular replacement in the head and neck cancer patient but should be reserved for lateral defects. For anterior reconstructions, even in patients with locally advanced disease, free-tissue transfer of composite osteocutaneous flaps is the reconstructive method of choice.     

Quantitatively Imaging Chromosomes by Correlated Cryo-Fluorescence and Soft X-Ray Tomographies

Soft x-ray tomography (SXT) is increasingly being recognized as a valuable method for visualizing and quantifying the ultrastructure of cryopreserved cells. Here, we describe the combination of SXT with cryogenic confocal fluorescence tomography (CFT). This correlative approach allows the incorporation of molecular localization data, with isotropic precision, into high-resolution three-dimensional (3-D) SXT reconstructions of the cell. CFT data are acquired first using a cryogenically adapted confocal light microscope in which the specimen is coupled to a high numerical aperture objective lens by an immersion fluid. The specimen is then cryo-transferred to a soft x-ray microscope (SXM) for SXT data acquisition. Fiducial markers visible in both types of data act as common landmarks, enabling accurate coalignment of the two complementary tomographic reconstructions. We used this method to identify the inactive X chromosome (Xi) in female v-abl transformed thymic lymphoma cells by localizing enhanced green fluorescent protein-labeled macroH2A with CFT. The molecular localization data were used to guide segmentation of Xi in the SXT reconstructions, allowing characterization of the Xi topological arrangement in near-native state cells. Xi was seen to adopt a number of different topologies with no particular arrangement being dominant.     

Quantitatively Imaging Chromosomes by Correlated Cryo-Fluorescence and Soft X-Ray Tomographies

Soft x-ray tomography (SXT) is increasingly being recognized as a valuable method for visualizing and quantifying the ultrastructure of cryopreserved cells. Here, we describe the combination of SXT with cryogenic confocal fluorescence tomography (CFT). This correlative approach allows the incorporation of molecular localization data, with isotropic precision, into high-resolution three-dimensional (3-D) SXT reconstructions of the cell. CFT data are acquired first using a cryogenically adapted confocal light microscope in which the specimen is coupled to a high numerical aperture objective lens by an immersion fluid. The specimen is then cryo-transferred to a soft x-ray microscope (SXM) for SXT data acquisition. Fiducial markers visible in both types of data act as common landmarks, enabling accurate coalignment of the two complementary tomographic reconstructions. We used this method to identify the inactive X chromosome (Xi) in female v-abl transformed thymic lymphoma cells by localizing enhanced green fluorescent protein-labeled macroH2A with CFT. The molecular localization data were used to guide segmentation of Xi in the SXT reconstructions, allowing characterization of the Xi topological arrangement in near-native state cells. Xi was seen to adopt a number of different topologies with no particular arrangement being dominant.     

The structure of FtsZ filaments in vivo suggests a force-generating role in cell division

In prokaryotes, FtsZ (the filamentous temperature sensitive protein Z) is a nearly ubiquitous GTPase that localizes in a ring at the leading edge of constricting plasma membranes during cell division. Here we report electron cryotomographic reconstructions of dividing Caulobacter crescentus cells wherein individual arc-like filaments were resolved just underneath the inner membrane at constriction sites. The filaments' position, orientation, time of appearance, and resistance to A22 all suggested that they were FtsZ. Predictable changes in the number, length, and distribution of filaments in cells where the expression levels and stability of FtsZ were altered supported that conclusion. In contrast to the thick, closed-ring-like structure suggested by fluorescence light microscopy, throughout the constriction process the Z-ring was seen here to consist of just a few short (approximately 100 nm) filaments spaced erratically near the division site. Additional densities connecting filaments to the cell wall, occasional straight segments, and abrupt kinks were also seen. An 'iterative pinching' model is proposed wherein FtsZ itself generates the force that constricts the membrane in a GTP-hydrolysis-driven cycle of polymerization, membrane attachment, conformational change, depolymerization, and nucleotide exchange.     

Rigid fixation of internal orbital fractures

When large portions of the internal orbit are destroyed (two to four walls), standard bone-grafting techniques for immediate and late orbital reconstruction may not yield predictable eye position. Critical bone support is most often deficient inferomedially. CT analysis of orbital volume in cases where eye position was unsatisfactory reveals that displacement of bone grafts is one mechanism of the unsatisfactory result. Other mechanisms include undercorrection and bone-graft resorption. In order to minimize postoperative bone-graft displacement, titanium implants were used to span large defects in the internal orbit to provide a platform for bone-graft support. Twenty-six implants were placed in immediate and 12 were placed in late orbital reconstructions. More reliable bone-graft position resulted. Two late infections have occurred resulting in implant removal in a 3-year period.     

Precise two-dimensional D-bar reconstructions of human chest and phantom tank via sinc-convolution algorithm

ABSTRACT: BACKGROUND: Electrical Impedance Tomography (EIT) is used as a fast clinical imaging technique formonitoring the health of the human organs such as lungs, heart, brain and breast. Eachpractical EIT reconstruction algorithm should be efficient enough in terms of convergencerate, and accuracy. The main objective of this study is to investigate the feasibility of preciseempirical conductivity imaging using a sinc-convolution algorithm in D-bar framework. METHODS: At the first step, synthetic and experimental data were used to compute an intermediate objectnamed scattering transform. Next, this object was used in a 2-day integral equation whichwas precisely and rapidly solved via sinc-convolution algorithm to find the square root of theconductivity for each pixel of image. For the purpose of comparison, multigrid and NOSERalgorithms were implemented under a similar setting. Quality of reconstructions of syntheticmodels was tested against GREIT approved quality measures. To validate the simulationresults, reconstructions of a phantom chest and a human lung were used. RESULTS: Evaluation of synthetic reconstructions shows that the quality of sinc-convolutionreconstructions is considerably better than that of each of its competitors in terms ofamplitude response, position error, ringing, resolution and shape-deformation. In addition, theresults confirm near-exponential and linear convergence rates for sinc-convolution andmultigrid, respectively. Moreover, the least degree of relative errors and the most degree oftruth were found in sinc-convolution reconstructions from experimental phantom data.Reconstructions of clinical lung data show that the related physiological effect is wellrecovered by sinc-convolution algorithm. CONCLUSIONS: Parametric evaluation demonstrates the efficiency of sinc-convolution to reconstruct accurateconductivity images from experimental data. Excellent results in phantom and clinicalreconstructions using sinc-convolution support parametric assessment results and suggest thesinc-convolution to be used for precise clinical EIT applications.     

Chromosome arrangements in human fibroblasts at mitosis

Summary The positions of the centromeres of all 46 human chromosomes were analysed in three dimensional reconstructions of electron micrographs of 10 serially sectioned unpretreated human male fibroblast cells. The reconstructions show that the spatial positioning of the chromosomes during division is not random. The centromeres were arranged on a metaphase plate that was ellipsoidal and that tended to be flat. The distance of centromeres from the centre of the mitotic figure was correlated with chromosome size; small chromosomes tended to be central in all the metaphases. Large chromosomes were more peripheral, especially in cells that were more advanced in mitosis. Thus, there is a tendency for larger chromosomes to move outwards as metaphase advances. In many cells, the A group centromeres were overdispersed, whereas G group centromeres tended to be clustered. The acrocentric chromosomes (D and G groups) also tended to be clustered when analysed together, probably reflecting associations in nucleoli at the previous interphase. The results show that chromosome disposition is non-random and that it changes during division.     

Identification of free coated pinocytic vesicles in Swiss 3T3 cells.

Whether or not free coated vesicles are involved during internalization of ligands bound to the receptors of coated pits is controversial. Free coated vesicles cannot be identified with certainty in random individual thin sections - reconstructions based on consecutive thin sections are required. The thickness of the sections determines the reliability of such reconstructions. In the present study, serial section electron microscopy was applied to Swiss 3T3 cells and the topographical resolution yielded by 80 nm and 20 nm sections was compared. Swiss 3T3 cells in monolayer at 37 degrees C were exposed for 5 min to cationized ferritin (CF) which is a marker of pinocytic vesicles. Subsequently the cells were fixed, pelleted and further processed for electron microscopy. The results showed that reconstructions of coated CF-labeled structures based on consecutive sections of an average thickness of approximately 80 nm could not be performed with certainty. A substantial fraction (25%) of the examined profiles appeared to be free vesicles, but narrow surface connections could easily have been missed in these thick sections. The series of the much thinner 20 nm sections provided a better resolution allowing the narrowest surface connections to be identified. Accordingly, the number of truly free, coated vesicles was much lower than the number of apparently free vesicles in the thick sections. However, free coated vesicles labeled with CF were identified in the consecutive 20 nm sections (4% of the examined profiles).     

Subject-specific body segment parameters' estimation using biplanar X-rays: a feasibility study

In order to improve the reliability of children's models, the aim of this study was to determine the subject-specific masses and 3D locations of the centres of mass (CoM) of body segments using biplanar X-rays. Previous methods, validated on upper leg segments, were applied to the whole body. Six children and six adults were studied. The low-dose X-ray system EOS(?) was used to simultaneously get head-to-foot biplanar X-rays in the upright position. Specific methods were used to get 3D reconstructions of bones and body shape. The densities from the literature were used to define the masses. To assess the accuracy of the reconstructions, a force plate was used to compare the mass and the projection of the CoM. A mean distance of 4.5?mm between the measured and the calculated projections of the CoM was found. The mean error between the estimated and the actual body mass was 2.6%. Such a method will be useful in obtaining the body segment parameters in children, hard to obtain using direct measurement techniques.     

The accuracy of DLT extrapolation in three-dimensional film analysis

An analysis of errors arising from the Direct Linear Transformation (DLT) approach to three-dimensional reconstructions from two-dimensional images has been undertaken, the principal factor studied being the number and distribution of control points used in the calibration procedure. Significantly increased error was found to be associated with extrapolation to unknown points outside the control point distribution space. Differences in accuracy between two camera position set-ups and 11 vs 12 DLT parameter solutions were also examined.     

Distribution of microtubules during centriole separation in rat kangaroo (Potorous) cells.

The distribution of microtubule profiles during separation of the centriole duplexes in prophase of rat kangaroo cells (PTK2W) has been investigated by serial section reconstructions. Separation begins with a reduction of the interphase microtubular network which radiates from the region of the duplex to all parts of the cell. As duplexes separate, microtubular elements appear between the duplexes. However, most microtubular elements extend oblique to the axis of separation rather than aligned along the axis of separation. Few microtubules are seen extending directly between duplexes. Longer profiles are seen as separation continues. Prior to the completion of separation, microtubules coalesce to form a discrete band often juxtaposed on the nuclear membrane. Subsequently, the duplexes reach their final position and nuclear envelope dissolution is initiated.     

A multimodal system for reconstruction and quantification of neurologic structures

The utility of computers and computer graphics as aids in the study of nervous system architecture is growing. However, modern histologic, immunocytologic and biochemical methods for revealing the underlying microarchitecture and macroarchitecture of the nervous system yield data formats requiring disparate computer acquisition, analysis and display approaches, capable of spanning many orders of magnitude of scale. This paper describes the Image Graphics Laboratory data acquisition, processing and display system, whose various components and programs may be used singly or in concert to enable definition of various tissue properties at different levels of resolution and integration. Examples are given of the system's use in light microscopic two-dimensional and three-dimensional reconstructions, autoradiographic reconstructions, reconstructions from projected images, reconstructions of impregnated cells (e.g., whole neurons) and peripheral nerve image analysis.     

Nucleoprotein-RNA orientation in the measles virus nucleocapsid by three-dimensional electron microscopy

Recombinant measles virus nucleoprotein-RNA (N-RNA) helices were analyzed by negative-stain electron microscopy. Three-dimensional reconstructions of trypsin-digested and intact nucleocapsids coupled to the docking of the atomic structure of the respiratory syncytial virus (RSV) N-RNA subunit into the electron microscopy density map support a model that places the RNA at the exterior of the helix and the disordered C-terminal domain toward the helix interior, and they suggest the position of the six nucleotides with respect to the measles N protomer.     

3D reconstruction of the ribs from lateral and frontal X-rays in comparison to 3D CT-scan reconstruction

Subject-specific three-dimensional (3D) reconstructions of the ribs can be obtained from biplanar X-rays. The goal of this study was to evaluate the accuracy and the inter-observer reproducibility of this technique in comparison to CT-scan reconstructions. CT scans and biplanar X-rays were obtained from 50 ribs (from three cadaveric rib cages). Three experienced experimenters reconstructed each rib from biplanar X-rays. Morphometric parameters were then computed from the rib midlines. Differences were computed between parameters obtained from the 3D reconstructions based on biplanar X-rays and from CT scans. The accuracy was computed as the mean of this difference for the 50 ribs from all three experimenters. The inter-observer variability was assessed using the coefficient of variation (CV) between the three observers. The CT-scan reconstructions were considered to be the gold standard in spite of their limitations for rib reconstructions. According to the different linear parameters, the accuracy of the reconstructions was found to be between -6mm (-2%) and 3mm, (4%). The accuracy of the current method was close to that of CT-scan reconstructions. The inter-observer variability was between 3% and 6%. Frontal and lateral X-rays are commonly obtained clinically, so 3D reconstructions can be used without increased radiation exposure to the patient.     

Three-dimensional reconstruction of whole cells of Streptococcus faecalis from thin sections of cells.

A new ultrastructural technique has been developed to study the geometry of cell wall assembly in Streptococcus faecalis, which is believed to occur between pairs of raised bands located on the organism's surface. Three-dimensional reconstructions of these new regions of envelope growth are produced from the mathematical rotation (around a central axis) of various measurements taken from central, longitudinal thin sections of cells. These reconstructions can be used to calculate the surface area and volume of the septal and peripheral walls that were supposedly present in any given cell before sectioning. In an accompanying paper, it is shown how such surface and volume estimations, coupled with other measurements of length, thickness, and curvature, can be used to characterize a cycle of envelope growth in this organism. The validity of the assumptions used to reconstruct cells by rotation and the possible sources of error in using this technique are discussed.     

Movement of Cell Organelles and the Geotropic Curvature in Roots of Norway Spruce (Picea abies)

The geotropic development in roots of Norway spruce [(Picea abies (L.)] H. Karst, has been followed by light and electron microscopy and compared with the movement of cell organelles (statoliths) in the root cap cells. The geotropic curvature develops in two phases: (a) an initial curvature in the root cap region, which results in an asymmetry in the extreme root tip and which appears after about 3 h stimulation in the horizontal position; and (b) the geotropic curvature in the basal parts of the root tip, which after 8 h is distributed over the entire elongation zone. A graphic extrapolation, based on measurements of the root curvatures after various stimulation periods, indicates a presentation time in the range of 8 to 10 min. The root anatomy and ultrastructure have been examined in detail in order to obtain information as to which organelles may act as gravity receptors. The root cap consists of a central core (columella) distinct from the peripheral part. The core contains three to four rows of parenchymatic cells each consisting of 15 to 18 storeys of statocyte cells with possibly mobile cell organelles. Amyloplasts and nuclei have been found to be mobile in the root cap cells, and the movement of both types of organelles has been followed after inversion of the seedlings and stimulation in the horizontal position for various periods of time at 4°C and 21°C. Three-dimensional reconstructions of spruce root cap cells based on serial sectioning and electron microscopy have been performed. These demonstrate that the endoplasmic reticulum (ER)-system and the vacuoles occupy a considerable part of the statocyte cell. For this reason the space available for free movement of single statolith particles is highly restricted.     

Deutocerebrum of the cockroach Blaberus craniifer burm. Quantitative study and automated identification of the glomeruli

The glomerular organization of the hemideutocerebrum is analyzed quantitatively, using only spatial position, in four individuals (eight hDTCs) after a visual identification of glomeruli on graphic reconstructions. In order to assess directly the invariance of the neuropil in an insect brain the following is done: (1) The position of each glomerulus is compared to the position A′ it should occupy if the hDTCs were identical. It is shown that in 80 and 71% of the cases, respectively, intra- and interindividual comparisons in the studied glomerulus is the nearest to A′. (2) The actual position is equally compared to the theoretical location A″ each glomerulus should occupy if its absolute position could be changed but not its relative position with respect to its neighbors. The calculation of A″ is based on process (3). In 86 and 80% of the cases, respectively, it is found that the actual position is that which is nearest to A″. (3) An automated identification process, based on absolute and relative locations, but completely independent of visual identification, is described. It allows the identification of 77 and 74% of glomeruli, respectively. These matchings are in 96 and 90% of the cases identical to the visual matchings. The location predictability of most glomeruli is discussed to show the existence, nature, and limits of the hemideutocerebral invariance.     

Comparison of identified mitral and tufted cells in freely breathing rats: I. Conduction velocity and spontaneous activity

The spontaneous activity and impulse conduction velocities of mitral and tufted cells were compared in the entire main olfactory bulb of freely breathing, anesthetized rats. Single units in the mitral cell body layer (MCL) and external plexiform layer (EPL) were identified by antidromic activation from the lateral olfactory tract (LOT), electrode track reconstructions based on dye marking, and the waveform of LOT-evoked field potentials. Using the track reconstructions, EPL units were further subdivided into glomerular border (GB) and not at the glomerular border (notGB) cells. For conduction velocity, significant differences were only found between MCL and GB units and not between MCL and all EPL units or between MCL and notGB units. For spontaneous activity, no significant differences were found between the different unit groups regarding the mean, maximum, or relative maximum rate per 100-ms bin. By contrast, they showed a differential modulation of their firing activity by respiration. GB but not notGB units had a significantly higher mean rate during the respiratory cycle than MCL units with significantly more activity during inspiration. Thus, mitral and tufted cells are similar in their impulse conduction velocity and spontaneous activity, though the more superficially placed GB cells exhibit differences. A comparison of odor responses in these cell types in the companion paper also points to differences between mitral and superficial projection tufted cells.