Biomedical subjects
B Carragher
Publications and source records attributed to B Carragher.
Re-evaluation of the structural organization of neuritic plaques in Alzheimer's disease.
We re-examined the relationship among synaptic pathology, subcellular abnormalities within neurites in the plaques and beta-amyloid deposits of Alzheimer's disease (AD) using laser confocal imaging and computer-aided serial section reconstruction techniques. Analysis of serial optical sections of neuritic plaques double-immunolabeled for anti-beta-amyloid/anti-tau-2 revealed that 35% of this type of plaque contained a dense amyloid core with clusters of peripheral abnormal neurites. The other 65% were without a dense core and were mainly composed of abundant abnormal neuritic clusters with bundles of amyloid distributed throughout the neuritic plaque. While two-dimensional (2-D) analysis of the plaques has suggested that neurites are distributed in the plaque periphery with beta-amyloid localized in its center, serial section analysis showed the opposite arrangement can also be true. Three-dimensional (3-D) reconstructions of serial optical sections showed that the tau-positive tortuous axons clustered in the neuritic plaques were often continuous with synaptophysin-positive distended terminals. Analysis of electron micrographs from serial sections showed continuity among the different segments of the neurites. Further analysis of the computer generated 3-D reconstructed neuritic plaques (both from serial electron micrographs and serial optical sections), viewed as continuous rotating loops, confirmed that a great majority of the plaque volume was occupied by the clustered and continuous abnormal neurites, while the amyloid fibrils were compressed and displaced to the periphery of the plaque. The 3-D imaging of the neuritic plaques in AD suggests a more widespread and active neuritic damage than that predicted from 2-D observations.(ABSTRACT TRUNCATED AT 250 WORDS)
Three-dimensional analysis of the relationship between synaptic pathology and neuropil threads in Alzheimer disease.
Recent studies have shown that the Alzheimer disease (AD) neocortex is characterized by a loss of large neurons, the presence of dilated terminal axons, widespread loss of synapses, and a disruption of the dendritic cytoskeleton which is manifested as Tau immunoreactive threads. In the present study we have investigated the relationship between synaptic and dendritic abnormalities in the neocortex of Alzheimer patients and examined the extent to which these structural alterations correlate with the severity of cognitive impairment in AD. Quantitative neuroanatomical data were obtained from immunofluorescence-labeled specimens using a laser-scanning confocal microscope, computer-assisted image processing and serial section reconstruction techniques. We found that the AD cases showed a 34% loss in the number of presynaptic terminals per 100 square (sq) microns, many of which showed structural abnormalities. The AD neuropil had an average of 10 +/- 7 dendritic threads per 1,000 sq microns, with the average thread measuring 2 sq microns. Severe AD cases had thicker threads compared with mild to moderate AD cases. Three-dimensional analysis showed clustering of synapses around threads, as well as presynaptic boutons apposed to dendritic neuropil threads. Statistical analysis showed that the strongest correlation was between synapse density and Blessed score of cognitive impairment. Thread counts did not correlate with either but were correlated with tangle counts. Stepwise multiple regression analysis showed that tangle counts, but not threads, strengthened the correlation between Blessed score and synapses. We conclude that synaptic damage may precede dendritic thread and tangle formation, and that threads do not necessarily induce synaptic pathology. Instead, dendrite sprouting in the denervated regions could be associated with increased accumulation of cytoskeletal proteins observed in the dendritic threads.
On the assembly of sickle hemoglobin fascicles.
Deoxyhemoglobin S fibers associate into bundles, or fascicles, that subsequently crystallize by a process of alignment and fusion. We have used electron microscopy to study the formation of fascicles and the changes in fiber packing that occur during the conversion of fascicles to crystals. The first event in crystallization involves fibers forming fascicles that are initially small and poorly ordered but, with time, become progressively larger and more highly ordered. After six to eight hours, the fibers in a fascicle form a crystalline lattice. The three-dimensional unit cell parameters of this lattice are a = 1300 A, b = 365 A, and c = 210 A (the a axis is parallel to the fiber axis). Fibers have an elliptical cross-section whose major and minor axes are 250 A and 185 A, respectively. When projected on to the unit cell vectors, these dimensions are 210 A and 155 A, so the unit cell dimension of 365 A implies that there are two fibers per unit cell. Theoretically, fibers could pair so that each member of the unit cell is oriented in the same direction (parallel) or opposite directions (antiparallel). Fourier transforms of electron micrographs (or models) cannot distinguish between these alternatives, since the two arrangements produce very similar intensity distributions. The orientation of the fibers was determined from cross-sections of the fascicles in which the fibers are seen end-on. In this view the images of the fibers are rotationally blurred because the fibers twist 30 degrees to 40 degrees about their helical axis through the 300 A to 400 A thick section. We have been able to remove the rotational blur from each of the fibers in the unit cell using the procedures described by Carragher et al. The deblurred images of the two fibers in the unit cell are related by mirror symmetry. This relationship means that the fibers are antiparallel. These observations suggest that crystallization of fibers in fascicles is mediated by assembly of the fibers into antiparallel pairs that contain equal numbers of double strands running in each direction.
Structural analysis of polymers of sickle cell hemoglobin. I. Sickle hemoglobin fibers.
The structure of fibers of deoxyhemoglobin S has been under investigation for several years and a number of different models have been proposed for the arrangement of molecules within the particles. We have used reconstruction and modeling techniques in our analysis of these structures. Several new approaches have been employed in this analysis in order to provide improved estimates of the co-ordinates, pairing, and polarity of the hemoglobin S molecules. Fibers have a variable pitch and, in order to minimize distortions in the reconstructed density maps associated with these variations in pitch, we have developed an iterative procedure to measure the instantaneous pitch and have modified the reconstruction algorithm to incorporate the measured values. This procedure improves the accuracy with which the hemoglobin S molecules can be located in the density maps. Furthermore, the determination of the instantaneous pitch allows us to measure directly the rotation of the individual hemoglobin molecules. These measurements are in excellent agreement with the values predicted using a random angular walk model (as originally proposed for F-actin) to describe the variable pitch. The reconstructions confirm that the fiber consists of 14 strands of hemoglobin S arranged in a hexagonally shaped cross-section. We have determined the pairing of the molecules to form double strands directly from the density maps by identifying the molecules that have intermolecular distances that conform to those of double strands in the Wishner-Love crystal. The seven double strands identified in this manner are consistent with the strand pairings proposed by Dykes et al. (1979) rather than the alternate pairings proposed by Rosen & Magdoff-Fairchild (1985). In addition, we have for the first time determined the polarity of the double strands directly from the reconstruction data. This was achieved using a procedure that amounts to essentially "dissecting" individual double strands from the reconstructed density maps so that their density distribution could be examined independently of the neighboring double strands. Knowledge of the relative polarities of the double strands is essential for determining the intermolecular interactions that stabilize the fiber.
Structural analysis of polymers of sickle cell hemoglobin. II. Sickle hemoglobin macrofibers.
Sickle cell hemoglobin macrofibers are an important intermediate in the low pH crystallization pathway of deoxygenated hemoglobin S that link the fiber to the crystal. Macrofibers are a class of helical particles differing primarily in their diameters but are related by a common packing of their constituent subunits. We have performed three-dimensional reconstructions of three types of macrofibers. These reconstructions show that macrofibers are composed of rows of Wishner-Love double strands in an arrangement similar to that in the crystal. We have measured the orientation and co-ordinates of double strands in macrofibers using cross-correlation techniques. In this approach, the electron density projections of double strands calculated from the known high-resolution crystal structure are compared with regions along the length of the particles in which the distinct pattern of double strands in c-axis projection may be observed. Contrary to assertions by Makinen & Sigountos (1984), our results unambigously demonstrate that adjacent rows of double strands in macrofibers are oriented in an antiparallel manner, as in the Wishner-Love crystal. Adjacent rows of antiparallel double strands are displaced along the helical axis relative to their co-ordinates in the crystal. Electron density models of macrofibers based on the crystallographic structure of the sickle hemoglobin double strand are in good agreement with the projections of macrofibers observed in electron micrographs. We have studied the structure of a closely related crystallization intermediate, the sickle hemoglobin paracrystal. The arrangement of double strands in paracrystals is similar to that in Wishner-Love crystals, except that they are displaced along the a-axis of the crystal. Measurements of the double strand co-ordinates reveal that the distribution of strand positions is bimodal. These results further establish the close structural relationship between macrofibers and paracrystals as intermediates in the crystallization of deoxygenated sickle hemoglobin.
Structural analysis of polymers of sickle cell hemoglobin. III. Fibers within fascicles.
We have examined the structure of hemoglobin S fibers, which are associated into large bundles, or fascicles. Electron micrographs of embedded and cross-sectioned fascicles provide an end-on view of the component fibers. The cross-sectional images are rotationally blurred as a result of the twist of the fiber within the finite thickness of the section. We have applied restoration techniques to recover a deblurred image of the fiber. The first step in this procedure involved correlation averaging images of cross-sections of individual fibers in order to improve the signal-to-noise ratio. The rotationally blurred image was then geometrically transformed to polar co-ordinates. In this space, the rotational blur is transformed into a linear blur. The linearly blurred image is the convolution of the unblurred image and a point spread function that can be closely approximated by a square pulse. Deconvolution in Fourier space, followed by remapping to Cartesian co-ordinates, produced a deblurred image of the original micrograph. The deblurred images indicate that the fiber is comprised of 14 strands of hemoglobin S. This result provides confirmation of the fiber structure determined using helical reconstruction techniques and indicates that the association of fibers into ordered arrays does not alter their molecular structure.
The reconstruction of helical particles with variable pitch.
In the reconstruction of helical particles, it is normally assumed that translation along the length of a particle is coupled to rotation about its axis. This assumption is not valid for particles whose pitch varies along the particle length (e.g. actin, HbS fibers), and application of the usual algorithms results in significant errors in both the shape and coordinates of subunits in the reconstructed density map. We have developed an iterative procedure for reconstructing particles with variable pitch. The goal of this procedure is to obtain an accurate estimate of the local pitch of the particle which can then be incorporated into the reconstruction algorithm. This involves synthesis of trial model structures which have constant pitch. The local pitch is derived from a cross-correlation analysis between these trial models and the variable pitch particles. The constant pitch models are constructed using coordinates measured from the reconstructed density maps. Each iteration of the procedure provides an improved estimate of the pitch which is incorporated into the succeeding iteration. The fidelity of the reconstruction is determined from cross-correlation between the original micrograph and a variable pitch model. The iterations are continued until the cross-correlation coefficient between the variable pitch model and the micrograph of the particle is maximized. The implementation of the iterative procedure is described and its behavior is evaluated using model structures which incorporate variations in pitch similar to those actually occurring in sickle hemoglobin fibers. The results indicate that the iterative reconstruction procedure considerably reduces the errors associated with constant pitch reconstructions. These tests provide a basis for applying this procedure in the structural analysis of micrographs of helical particles which display variable pitch. Application to sickle hemoglobin fibers resulted in an improvement in the accuracy with which the hemoglobin S molecules can be located in the density maps.
The three-dimensional structure of sickle hemoglobin macrofibers.
The crystallization of deoxygenated sickle cell hemoglobin (HbS) at pH less than 6.5 is preceded by the formation of HbS macrofibers. In stirred solutions, macrofibers appear several hours after the formation of fibers and subsequently aggregate and crystallize. Like fibers, macrofibers are long helical structures consisting of alternating narrow and wide regions. Macrofibers have a pitch of 10,000 A and a diameter averaging 500 A. Here we present a three-dimensional structural analysis of HbS macrofibers. Image reconstructions of these particles show that they consist of 5 antiparallel rows with 8, 11, 12, 11, and 8 double strands per row. Double strands in the outer two rows are asymmetrically disposed resulting in "notches" in opposite corners of the particle cross section. The notches produce characteristic features in electron micrographs of macrofibers. Fourier-filtered images of macrofibers have been produced which reveal their structure in considerable detail. Computed Fourier transforms of macrofibers exhibit spacings of 64 A, 185 A, and 53 A similar to those in the Wishner-Love crystal. A cross-correlation analysis between filtered macrofiber images and electron density projections of Wishner-Love double strands establishes that double strands in adjacent rows are antiparallel. This analysis also shows that the macrofiber rows are displaced axially by differing amounts depending on their distance from the helical axis. Using this data, we have computed a three-dimensional electron density model of the macrofiber based on atomic hemoglobin coordinates. This model reproduces in detail the many features revealed in Fourier-filtered macrofiber images.
The restoration of electron micrographs blurred by drift and rotation.
We have investigated the restoration of electron micrographs exhibiting blurring due to drift and rotation. Blurring due to drift arises in micrographs taken of a specimen which is moving relative to the image plane. A related problem is that of rotational blurring which arises in micrographs of thin sections of helical particles viewed in cross section. The twist of the particle within the finite thickness of the section causes the image to appear rotationally blurred about the helical axis. Restoration algorithms were evaluated by applying them to the restoration of blurred model images degraded by additive Gaussian noise. Model images were also used to investigate how an incorrect estimate of the point spread function describing the blur would effect the restoration. Images were, if necessary, geometrically transformed to a space in which the point spread function of the blur can be considered as linear and space invariant as, under these conditions, the restoration algorithms are greatly simplified. In the case of the rotationally blurred images this procedure was accomplished by transforming the image to polar coordinates. The restoration techniques were successfully applied to blurred micrographs of bacteriophage T4 and crystals of catalase. The quality of the restoration was judged by comparisons of the restored images to undegraded images. Application to micrographs of rotationally blurred cross sections of helical macrofibers of sickle hemoglobin resulted in a reduction in the amount of rotational blurring.
Advances in computational image processing for microscopy.
The field of electron microscopy applied to structural biology has made significant progress in the last 5 years, with many labs attempting the solution of ever more challenging problems. This work has demanded significant new resources for the computational analysis of the data collected from the microscope. The purpose of this paper is to review briefly the state of the art in image processing of microscope data and to serve as an introduction to this special issue of the journal.
Helical processing using PHOELIX.
We describe here a set of procedures and algorithms for helical processing that we refer to as the PHOELIX package. The package was developed to provide a time-efficient and semiautomated method for determining a three-dimensional density map from a specimen with helical symmetry. The procedures which are part of PHOELIX are drawn from the original MRC helical processing suite with extensions principally developed using the SUPRIM image processing package. The package in its current form has been optimized for the processing of actomyosin filaments but has been modified and applied to other helical structures.