M. Cynthia Goh

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.

A quantitative diffraction-based sandwich immunoassay.

It is shown that diffraction-based sensing can be enhanced for diagnostic purposes through the use of a secondary label. The limit of detection for anti-rabbit IgG was reduced more than 40-fold by using a gold-conjugated secondary antibody. The response to secondary antibody binding was linear for concentrations from 25 to 500 ng/ml of anti-rabbit IgG, suggesting that quantitative determinations can be readily done. Moreover, the binding of the secondary antibody was observed as soon as 1 min after its introduction to the surface-bound primary complex.

Fibrous Long Spacing Collagen Ultrastructure Elucidated by Atomic Force Microscopy

Fibrous long spacing collagen (FLS) fibrils are collagen fibrils in which the periodicity is clearly greater than the 67-nm periodicity of native collagen. FLS fibrils were formed in vitro by the addition of alpha1-acid glycoprotein to an acidified solution of monomeric collagen and were imaged with atomic force microscopy. The fibrils formed were typically approximately 150 nm in diameter and had a distinct banding pattern with a 250-nm periodicity. At higher resolution, the mature FLS fibrils showed ultrastructure, both on the bands and in the interband region, which appears as protofibrils aligned along the main fibril axis. The alignment of protofibrils produced grooves along the main fibril, which were 2 nm deep and 20 nm in width. Examination of the tips of FLS fibrils suggests that they grow via the merging of protofibrils to the tip, followed by the entanglement and, ultimately, the tight packing of protofibrils. A comparison is made with native collagen in terms of structure and mechanism of assembly.

A statistically derived parameterization for the collagen triple-helix.

The triple‐helix is a unique secondary structural motif found primarily within the collagens. In collagen, it is a homo‐ or hetero‐tripeptide with a repeating primary sequence of (Gly‐X‐Y)n, displaying characteristic peptide backbone dihedral angles. Studies of bulk collagen fibrils indicate that the triple‐helix must be a highly repetitive secondary structure, with very specific constraints. Primary sequence analysis shows that most collagen molecules are primarily triple‐helical; however, no high‐resolution structure of any entire protein is yet available. Given the drastic morphological differences in self‐assembled collagen structures with subtle changes in assembly conditions, a detailed knowledge of the relative locations of charged and sterically bulky residues in collagen is desirable. Its repetitive primary sequence and highly conserved secondary structure make collagen, and the triple‐helix in general, an ideal candidate for a general parameterization for prediction of residue locations and for the use of a helical wheel in the prediction of residue orientation. Herein, a statistical analysis of the currently available high‐resolution X‐ray crystal structures of model triple‐helical peptides is performed to produce an experimentally based parameter set for predicting peptide backbone and Cβ atom locations for the triple‐helix. Unlike existing homology models, this allows easy prediction of an entire triple‐helix structure based on all existing high‐resolution triple‐helix structures, rather than only on a single structure or on idealized parameters. Furthermore, regional differences based on the helical propensity of residues may be readily incorporated. The parameter set is validated in terms of the predicted bond lengths, backbone dihedral angles, and interchain hydrogen bonding.

Investigating the ultrastructure of fibrous long spacing collagen by parallel atomic force and transmission electron microscopy

The ultrastructure of fibrous long spacing (FLS) collagen fibrils has been investigated by performing both atomic force microscopy (AFM) and transmission electron microscopy (TEM) on exactly the same area of FLS collagen fibril samples. These FLS collagen fibrils were formed in vitro from type I collagen and α1‐acid glycoprotein (AAG) solutions. On the basis of the correlated AFM and TEM images obtained before and after negative staining, the periodic dark bands observed in TEM images along the longitudinal axis of the FLS collagen fibril correspond directly to periodic protrusions seen by AFM. This observation is in agreement with the original surmise made by Gross, Highberger, and Schmitt (Gross J, Highberger JH, Schmitt FO, Proc Natl Acad Sci USA 1954;40:679‐688) that the major repeating dark bands of FLS collagen fibrils observed under TEM are thick relative to the interband region. Although these results do not refute the idea of negative stain penetration into gap regions proposed by Hodge and Petruska (Petruska JA, Hodge AJ. Aspects of protein structure. Ramachandran GN, editor. New York: Academic Press; 1963. p. 289–300), there is no need to invoke the presence of gap regions to explain the periodic dark bands observed in TEM images of FLS collagen fibrils. Proteins 2002;49:378–384.

Hierarchical assembly and the onset of banding in fibrous long spacing collagen revealed by atomic force microscopy

The mechanism of formation of fibrillar collagen with a banding periodicity much greater than the 67 nm of native collagen, i.e. the so-called fibrous long spacing (FLS) collagen, has been speculated upon, but has not been previously studied experimentally from a detailed structural perspective. In vitro, such fibrils, with banding periodicity of approximately 270 nm, may be produced by dialysis of an acidic solution of type I collagen and alpha(1)-acid glycoprotein against deionized water. FLS collagen assembly was investigated by visualization of assembly intermediates that were formed during the course of dialysis using atomic force microscopy. Below pH 4, thin, curly nonbanded fibrils were formed. When the dialysis solution reached approximately pH 4, thin, filamentous structures that showed protrusions spaced at approximately 270 nm were seen. As the pH increased, these protofibrils appeared to associate loosely into larger fibrils with clear approximately 270 nm banding which increased in diameter and compactness, such that by approximately pH 4.6, mature FLS collagen fibrils begin to be observed with increasing frequency. These results suggest that there are aspects of a stepwise process in the formation of FLS collagen, and that the banding pattern arises quite early and very specifically in this process. It is proposed that typical 4D-period staggered microfibril subunits assemble laterally with minimal stagger between adjacent fibrils. alpha(1)-Acid glycoprotein presumably promotes this otherwise abnormal lateral assembly over native-type self-assembly. Cocoon-like fibrils, which are hundreds of nanometers in diameter and 10-20 microm in length, were found to coexist with mature FLS fibrils.

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.

Demonstration of diffraction enhancement via Bloch surface waves in a-SiN:H multilayers

Using the excitation of a Bloch Surface Wave (BSW), we demonstrate a 45-fold diffraction enhancement for a protein grating printed on a-SiN:H multilayers. This may lead to a new generation of high sensitivity diffraction-based biosensors.