Peter Markiewicz

Sequential assembly of collagen revealed by atomic force microscopy

Most polymers which comprise biological filaments assemble by two mechanisms: nucleation and elongation or a sequential, stepwise process involving a hierarchy of intermediate species. We report the application of atomic force microscopy (AFM) to the study of the early events in the sequential or stepwise mode of assembly of a macromolecular filament. Collagen monomers were assembled in vitro and the early structural intermediates of the assembly process were examined by AFM and correlated with turbidimetric alterations in the assembly mixture. The assembly of collagen involved a sequence of distinctive filamentous species which increased in both diameter and length over the time course of assembly. The first discrete population of collagen oligomers were 1-2 nm in diameter (300-500 nm in length); at later time points, filaments approximately 2-6 nm in diameter (> 10 microns in length) many with a conspicuous approximately 67-nm axial period were observed. Occasional mature collagen fibrils with a approximately 67-nm axial repeat were found late in the course of assembly. Our results are consistent with initial end-to-end axial association of monomers to form oligomers followed by lateral association into higher-order filaments. On this basis, there appears to be at least two distinctive types of structural interactions (axial and lateral) which are operative at different levels in the assembly hierarchy of collagen.

Atomic force microscope tip deconvolution using calibration arrays

An algorithm for the deconvolution of the probe tips used with the atomic force microscope from the images of calibration grids made using very large scale integrated technology, which is useful in visualization of the tip before or after imaging for the elucidation of the condition of the stylus, is presented. This procedure described is of general applicability: it makes no assumptions about the tip geometry and requires no pretreatment of the data, such as filtering. Grids having circular depressions are shown to reproduce the geometries of circular and conical tips faithfully, but are limited when imaging those which are square pyramidal. Slight variations in the size and shapes of the depressions within a grid pattern limit the accuracy of the tip reconstruction.

Simulation of atomic force microscope tip–sample/sample–tip reconstruction

The image obtained with the atomic force microscope is a convolution of the tip and sample. A numerical algorithm which has been previously reported enables the removal of the tip geometry, hence exposing a more accurate picture of the sample. The efficacy of such a scheme is explored with the use of simulations of the tip–sample interaction using simple geometric considerations. Two examples that illustrate the limitations of image analysis by such procedures are presented.

Paired helical filaments are twisted ribbons composed of two parallel and aligned components: image reconstruction and modeling of filament structure using atomic force microscopy.

To study the structure of Alzheimer paired helical filaments (PHF) we examined isolated detergent-insoluble PHF using atomic force microscopy with image reconstruction. The reconstructed AFM images of Alzheimer PHF most closely resembled ribbon-like helices with thin edges. The presence of a conspicuous furrow in the PHF midline indicated that PHF were composed of two distinctive strands. Our present conception of the overall configuration of PHF is consistent with that proposed by Crowther and Wischik in 1985 but includes an essential component of the prevailing model: the presence of two strands. Thus, our new model of PHF structure, based on atomic force microscopy-derived data, indicates that the true structure of PHF is actually a hybrid of the prevailing PHF model and a thin helical ribbon.

Identifying locations on a substrate for the repeated positioning of AFM samples

Abstract A simple addition to the preparation of an AFM sample allows for the mapping of regions on the substrate. The technique makes use of copper locator grids placed under a translucent substrate such as mica or glass. This cost effective procedure allows one to reproducibly locate features over a sample surface area of approximately 7 mm 2 . Application of this procedure in locating a small group of spheres and in the annealing of a latex monolayer are shown. The procedure can also be of use in cases where the sample must be rotated or removed.

Identification and visualization of questionable regions in atomic force microscope images

An algorithm for the identification of areas that do not necessarily represent the true sample surface in atomic force microscope images is presented. These areas describe regions of the surface which might not have made contact with the probe tip during a raster scan, giving data which should be deemed questionable. Through the identification of these questionable data points, a more accurate picture of the sample can be obtained. The procedure is applied to several atomic force microscope images for the improvement of sample images and for obtaining tip information. While the algorithm is applicable to all such images, its sensitivity to noise reflects shortcomings in the assumption made in deconvolution.

Alzheimer paired helical filaments: a comparison with the twisted ribbon model.

To investigate if Alzheimer paired helical filaments (PHF) closely resemble twisted ribbons, as indicated by recent high-resolution ultrastructural studies, we compared physical models of twisted ribbons with electron microscopic images of PHF. Uranyl-acetate-stained, isolated PHF with one or two helical turns were compared with scale models of twisted ribbons with one and two helical turns rotated at different angles. The various rotations of the twisted ribbon model corresponded well with the different orientations of randomly dispersed PHF. The electron-dense regions of individual PHF turns previously thought to represent a cross-over site of paired filaments corresponded to the edge of the twisted ribbon when the ribbon was oriented perpendicular to the filament axis. These data indicate that the overall configuration of PHF is a twisted ribbon but does not exclude possible configuration restrictions due to an ordered arrangement of subunits.

FINE STRUCTURE OF ABNORMAL FILAMENTS ISOLATED FROM ALZHEIMER'S DISEASED BRAIN : AN APPLICATION OF ATOMIC FORCE MICROSCOPY

Abstract Alzheimers disease is characterized by the spontaneous assembly of a cytoskeletal protein into highly insoluble filamentous structures which appear, in conventional transmission electron microscopy (EM), as paired helical filaments (PHFs) or straight filaments (SFs). Recent examination of PHF ultrastructure using EM of vertical PtC replicas of isolated PHF indicate that PHFs more closely resemble twisted ribbons than helically wound pairs of filaments. To re-evaluate the fine structure of PHFs we isolated fractions highly enriched in PHF from Alzheimer diseased brains and characterized the fractions with an atomic force microscope. PHFs adhered to both mica and graphite surfaces without pre-treatment of the surfaces and remained fixed to the substrate even after repeated scanning. Using the AFM, PHFs appeared as twisted ribbons and had dimensions similar to those reported for vertically replicated PHF viewed with EM. Rare SFs were found admixed with typical twisted ribbons. These data indicate that high resolution imaging using both PtC replication and AFM give similar quantitative data with respect to PHF dimensions. We conclude that Alzheimer PHFs should be modelled as a twisted ribbon rather than the structure implied by its name.