Neil H. Thomson

Short cantilevers for atomic force microscopy

We have designed and tested a family of silicon nitride cantilevers ranging in length from 23 to 203 μm. For each, we measured the frequency spectrum of thermal motion in air and water. Spring constants derived from thermal motion data agreed fairly well with the added mass method; these and the resonant frequencies showed the expected increase with decreasing cantilever length. The effective cantilever density (calculated from the resonant frequencies) was 5.0 g/cm3, substantially affected by the mass of the reflective gold coating. In water, resonant frequencies were 2 to 5 times lower and damping was 9 to 24 times higher than in air. Thermal motion at the resonant frequency, a measure of noise in tapping mode atomic force microscopy, decreased about two orders of magnitude from the longest to the shortest cantilever. The advantages of the high resonant frequency and low noise of a short (30 μm) cantilever were demonstrated in tapping mode imaging of a protein sample in buffer. Low‐noise images were tak...

Direct Observation of One-Dimensional Diffusion and Transcription by Escherichia coli RNA Polymerase

The dynamics of nonspecific and specific Escherichia coli RNA polymerase (RNAP)-DNA complexes have been directly observed using scanning force microscopy operating in buffer. To this end, imaging conditions had to be found in which DNA molecules were adsorbed onto mica strongly enough to be imaged, but loosely enough to be able to diffuse on the surface. In sequential images of nonspecific complexes, RNAP was seen to slide along DNA, performing a one-dimensional random walk. Heparin, a substance known to disrupt nonspecific RNAP-DNA interactions, prevented sliding. These observations suggest that diffusion of RNAP along DNA constitutes a mechanism for accelerated promoter location. Sequential images of single, transcribing RNAP molecules were also investigated. Upon addition of 5 microM nucleoside triphosphates to stalled elongation complexes in the liquid chamber, RNAP molecules were seen to processively thread their template at rates of 1.5 nucleotide/s in a direction consistent with the promoter orientation. Transcription assays, performed with radiolabeled, mica-bound transcription complexes, confirmed this rate, which was about three times smaller than the rate of complexes in solution. This assay also showed that the pattern of pause sites and the termination site were affected by the surface. By using the Einstein-Sutherland friction-diffusion relation the loading force experienced by RNAP due to DNA-surface friction is estimated and discussed.

Biological applications of the AFM: From single molecules to organs

The application domains of the atomic force microscope have increased dramatically in recent years. We present a short review of the contributions of this microscope to biology. These are illustrated through the study of different samples, starting with the imaging of single molecules all the way up through the length scales, and ending with imaging of tissues. So that nonbiologists can appreciate the significance of these studies, special attention has been paid to a description of the samples and to point out the motivation of these studies and their implications for the field of medicine.

PROTEIN TRACKING AND DETECTION OF PROTEIN MOTION USING ATOMIC FORCE MICROSCOPY

Height fluctuations over three different proteins, immunoglobulin G, urease, and microtubules, have been measured using an atomic force microscope (AFM) operating in fluid tapping mode. This was achieved by using a protein-tracking system, where the AFM tip was periodically repositioned above a single protein molecule (or structure) as thermal drifting occurred. Height (z-piezo signal) data were taken in 1 - or 2-s time slices with the tip over the molecule and compared to data taken on the support. The measured fluctuations were consistently higher when the tip was positioned over the protein, as opposed to the support the protein was adsorbed on. Similar measurements over patches of an amphiphile, where the noise was identical to that on the support, suggest that the noise increase is due to some intrinsic property of proteins and is not a result of different tip-sample interactions over soft samples. The orientation of the adsorbed proteins in these preliminary studies was not known; thus it was not possible to make correlations between the observed motion and specific protein structure or protein function beyond noting that the observed height fluctuations were greater for an antibody (anti-bovine IgG) and an enzyme (urease) than for microtubules.

Methods for fabricating and characterizing a new generation of biomimetic materials

Bringing together current ideas in the fields of biomineralization and composite laminate materials, we have attempted to fabricate model materials that mimic abalone nacre through the rapid assembly of inorganic tablets, such as talc. Several physical methods were tested to aid the orientation of the talc tablets in fluid suspensions with a low percentage, 10% by dry weight, of organic binding material. The orientation of talc tablets in the synthesized composites was characterized by X-ray diffraction and scanning electron microscopy. The modulus of rupture of the materials was measured in a three-point bending test. We demonstrate that the alignment of tablets increases by the use of physical methods and from chemical surface treatment. Important factors to consider in making materials that mimic abalone nacre are discussed. Important factors to consider in making materials that mimic abalone nacre are discussed.

Oriented, Active Escherichia coli RNA Polymerase: An Atomic Force Microscope Study

Combining a system for binding proteins to surfaces (Sigal, G. B., C. Bamdad, A. Barberis, J. Strominger, and G. M. Whitesides. 1996. Anal. Chem. 68:490-497) with a method for making ultraflat gold surfaces (Hegner, M., P. Wagner, and G. Semenza. 1993. Surface Sci. 291:39-46 1993) has enabled single, oriented, active Escherichia coli RNA polymerase (RNAP) molecules to be imaged under aqueous buffer using tapping-mode atomic force microscopy (AFM). Recombinant RNAP molecules containing histidine tags (hisRNAP) on the C-terminus were specifically immobilized on ultraflat gold via a mixed monolayer of two different omega-functionalized alkanethiols. One alkanethiol was terminated in an ethylene-glycol (EG) group, which resists protein adsorption, and the other was terminated in an N-nitrilotriacetic acid (NTA) group, which binds the histidine tag through two coordination sites with a nickel ion. AFM images showed that these two alkanethiols phase-segregate. Specific binding of the hisRNAP molecules was followed in situ by injecting proteins directly into the AFM fluid cell. The activity of the hisRNAP bound to the NTA groups was confirmed with a 42-base circular single-stranded DNA template (rolling circle), which the RNAP uses to produce huge RNA transcripts. These transcripts were imaged in air after the samples were rinsed and dried, since RNA also has low affinity for the EG-thiol and cannot be imaged under the buffers we used.

Real‐time imaging of enzymatic degradation of starch granules by atomic force microscopy

Atomic force microscopy has been used to image wheat seed starch granules (cultivar Timmo) both in air and under water. In the aqueous environment, the enzyme α‐amylase was introduced into the solution cell of the microscope and its progressive attack on a starch granule was followed by collecting successive images of the granule. Each image was recorded over a time period of 105 s. The enzyme created a ‘‘pin‐hole’’ in the top of the granule, consistent with previous off‐line electron microscopy studies.

Scanning probe microscopes : Applications in cereal science

ABSTRACT Scanning probe microscopes (SPMs) are a family of related instruments which can produce high-resolution images of structures at the molecular and atomic levels. Their main impact so far has been in surface science, but they also have tremendous potential for the study of biomolecules. In this article we will briefly introduce the properties and principles of these instruments, and then discuss a range of preliminary studies from our laboratories in relation to future applications in cereal science.