Direct observation of microtubules with the scanning tunneling microscope

To observe surface topography of microtubules, we have applied scanning tunneling microscopy (STM), which can image metal and semiconductive surfaces with atomic resolution. Isolated microtubules fixed in 0.1% glutaraldehyde in reassembly buffer containing 0.8 M glycerol were imaged in air on a graphite substrate. The presence of microtubules in solution was verified by electron microscopy. At atmospheric pressure and room temperature, STM probing of both freeze-dried and hydrated microtubules reveals structures approximately 25 nm in width, consisting of longitudinal filaments about 4 nm in width. These structures match electron microscopy images of microtubules and their component protofilaments. Microtubules imaged by STM frequently appear buckled and semiflattened. Top-view shaded scans show what appear to be individual tubulin subunits within protofilaments. We believe these results represent the first direct STM observation of protein assemblies in which components can be identified. Although the microtubule image resolution described here is no better than that presently obtainable by other techniques (e.g., electron microscopy with freeze-drying, shadowing, and/or negative staining), it is significant that suitably prepared biomolecules may be sufficiently conductive and stable for STM imaging, which is ultimately capable of atomic resolution. Further development of STM technology, computer-enhanced image processing, and elucidation of optimal STM sample preparation indicate that STM and related applications will offer unique opportunities for the study of biomolecular surfaces.     

Charge contrast imaging of biomaterials in a variable pressure scanning electron microscope

Charge contrast imaging (CCI) is a dynamic phenomenon recently reported in insulating and semiconducting materials imaged with low vacuum or variable pressure scanning electron microscopes (SEM). Data presented in this paper illustrates that CCI can also be applied to biominerals and biological soft-tissues and that useful and unique structural information can be obtained from routine samples. Various resin-embedded samples were considered and example images from several different biomaterials are presented. Due to the diverse nature of samples that appear to exhibit charge contrast, this imaging technique has prospective application in a wide range of biological and biomedical research. This work represents the first application of CCI to biomaterials and in particular, highlights a new method for investigating the formation, structure and growth of biominerals.     

Imaging of cell membraneous and cytoskeletal structures with a scanning tunneling microscope

The first observation of unstained cell membraneous structures by a scanning tunneling microscope is reported. An adhesive preparation method was used for imaging human medulloblastoma cells from the cell line TE 671 and oocytes from the clawed toad Xenopus laevis. The images show filaments, stacks of molecules and hilly structures. The possible identify of the filamentous structures is discussed, although the observed structures cannot yet be fully characterized. The work suggests possible future experiments on various biological structures in their natural environment.     

High-resolution imaging using a novel atomic force microscope and confocal laser scanning microscope hybrid instrument: essential sample preparation aspects

The recent data explosion in global gene expression profiling and proteomics has resulted in a need to determine the mechanistic role of biomarker signatures in pathogenicity. Consequently, elaborate technologies are required to assess increasingly smaller sub-cellular compartments and constituents. We describe the development, evaluation and application of an efficient sample preparation methodology to facilitate coupled atomic force microscopy and confocal laser scanning microscopy (AFM–CLSM), providing a novel means of concurrent high-resolution structural and fluorescence imaging. Due to their fragile nature and nanoscale dimensions, filopodia were selected as a model to develop the procedure that maximised fluorescence response, while maintaining epithelial cell ultra-structure. Fixation with ultra-pure methanol-free formaldehyde coupled to quantum dot nanocrystal labelling proved to be vital in achieving high quality AFM–CLSM images. We demonstrated for the first time that filopodia have a “quilted” surface structure. Additionally, high ultra-structural ridges on the apical cell surface resolved by AFM corresponded to punctate moesin clusters, representing direct visualisation of moesin linkages between transmembrane proteins and the cytoskeleton. The capacity of this novel multi-modal imaging technique to probe topography, molecular composition and biophysical properties of ultra-structural features therefore provides unique information that will significantly contribute to our understanding of cellular structure–function relationships.     

Striving for atomic resolution in biomolecular topography: The scanning force microscope (SFM)

The invention in 1986 of scanning force microscopy (SFM) provided a new and powerful tool for the investigation of biological structures. SFM yields a three-dimensional view at nanometer resolution of the surface topography associated with biological objects. The potential for imaging either macromolecules or biomolecules and cells under native (physiological) conditions is currently being exploited to obtain functional information at the molecular level. In addition, the forces involved in individual bimolecular interactions are being assessed under static and dynamic conditions. In this report we focus on the imaging capability of the SFM. The rather broad spectrum of applications represented is intended to orient the prospective user of biological SFM.     

Imaging soft samples with the atomic force microscope: gelatin in water and propanol.

We have imaged mica coated with thin gelatin films in water, propanol, and mixtures of these two liquids by atomic force microscopy (AFM). The elastic modulus (Young's modulus) can be tuned from 20 kPa to more than 0.1 GPa depending on the ratio of propanol to water. The resolution is best in pure propanol, on the order of 20 nm, and becomes worse for the softer samples. The degradation in resolution can be understood by considering the elastic indentation of the gelatin caused by the AFM tip. This indentation becomes larger and thus the contact area becomes larger the softer the sample is. Therefore this study may be used to estimate the resolution to be expected with an AFM on other soft samples, such as cells. Nondestructive imaging was possible only by imaging at forces < 1 nN. This was difficult to achieve in contact mode because of drift in the zero load deflection of the cantilever, supposedly caused by temperature drift, but straightforward in tapping mode.     

The organization of the mammalian kinetochore: a scanning electron microscope study

Summary A procedure has been developed for scanning electron microscopy that enables the visualization of kinetochores along the surface of isolated chromosomes of the Indian muntjac. Indirect immunofluorescence and thin section analysis of the kinetochores of those isolated chromosomes verified that these structures retained in vivo composition and morphology during the isolation procedure. In scanning electron micrographs the outer surface of the outer kinetochore plate can be visualized as a series of fibers 25–30 nm in diameter that are arranged across the plate. These images are comparable to those obtained by whole mount transmission electron microscopic procedures (Rattner 1986) and are compatible with a model of the kinetochore in which chromatin fiber from the body of the chromosome extend to the outer kinetochore plate.     

Study of soft X-ray emission from Z-pinches with a complex atomic composition

Results are presented from experimental studies of Z-pinches produced by implosion of aluminum and tungsten cylindrical wire arrays in the Angara-5-1 facility. The electron temperature T _e and density n _e of the high-temperature pinch plasma have been determined by analyzing line emission from multicharged ions. For the same mass and radius of the array and the same number of wires in it, the intensity of line emission of H- and He-like Al ions from an imploded Al + W wire array containing even a small amount of tungsten (7 wt %) is one order of magnitude lower than that from an Al array. As the W content increases, the total soft X-ray (SXR) yield increases, while the duration of the SXR pulse decreases. For the 30% W content in the array, the power and duration of the SXR pulse are nearly the same as those recorded during the implosion of a W array with the same linear mass and radius and the same number of wires. Results are also presented from experiments with nested wire arrays in which the outer and inner shells were made of Al and W wires, respectively. It is found that, in this case, the effect of tungsten on the line emission of aluminum is much weaker than that in experiments with arrays in which tungsten and aluminum wires were placed in the same shell, even if the mass of the inner (tungsten) shell was larger than that of the outer (aluminum) one. At the same time, the inner W shell plays a significant role in the implosion dynamics of a nested wire array, reducing the duration of the SXR pulse and increasing the SXR power.     

Direct visualization of phosphorylase-phosphorylase kinase complexes by scanning tunneling and atomic force microscopy.

In skeletal muscle the activation of phosphorylase b is catalyzed by phosphorylase kinase. Both enzymes occur in vivo as part of a multienzyme complex. The two enzymes have been imaged by atomic force microscopy and the results compared to those previously found by scanning tunneling microscopy. Scanning tunneling microscopy and atomic force microscopy have been used to view complexes between the activating enzyme phosphorylase kinase and its substrate phosphorylase b. Changes in the size and shape of phosphorylase kinase were observed when it bound phosphorylase b.     

Measurement of the unstained biological sample by a novel scanning electron generation X-ray microscope based on SEM

We introduced a novel X-ray microscope system based on scanning electron microscopy using thin film, which enables the measurement of unstained biological samples without damage. An unstained yeast sample was adsorbed under a titanium (Ti)-coated silicon nitride (Si₃N₄) film 90 nm thick. The X-ray signal from the film was detected by an X-ray photodiode (PD) placed below the sample. With an electron beam at 2.6 kV acceleration and 6.75 nA current, the yeast image is obtained using the X-ray PD. The image is created by soft X-rays from the Ti layer. The Ti layer is effective in generating the characteristic 2.7-nm wavelength X-rays by the irradiation of electrons. Furthermore, we investigated the electron trajectory and the generation of the characteristic X-rays within the Ti-coated Si₃N₄ film by Monte Carlo simulation. Our system can be easily utilized to observe various unstained biological samples of cells, bacteria, and viruses.     

Determination of elastic moduli of thin layers of soft material using the atomic force microscope

We address three problems that limit the use of the atomic force microscope when measuring elastic moduli of soft materials at microscopic scales. The first concerns the use of sharp cantilever tips, which typically induce local strains that far exceed the linear material regime. We show that this problem can be alleviated by using microspheres as probes, and we establish the criteria for their use. The second relates to the common use of the Hertz contact mechanics model, which leads to significant errors when applied to thin samples. We develop novel, simple to use corrections to apply for such cases. Samples that are either bonded or not bonded to a rigid substrate are considered. The third problem concerns the difficulty in establishing when contact occurs on a soft material. We obtain error estimates for the elastic modulus resulting from such uncertainty and discuss the sensitivity of the estimation methods to error in contact point. The theoretical and experimental results are compared to macroscopic measurements on poly(vinyl-alcohol) gels.     

Scanning electron microscope histochemistry: The use of backscattered electrons to identify epidermal Langerhans cells in the scanning electron microscope

Summary Epidermal sheets reacted for ATPase were examined in the scanning electron microscope and images based on atomic number contrast formed by collecting the backscattered electrons. Langerhans cells lying within the tissue were revealed and could be related to surface structures by reference to conventional secondary electron images.     

A study of lignin formation at the molecular level by scanning tunneling microscopy.

A scanning tunneling microscope (STM) was used to observe the temporal formation and organization of dehydrogenative polymer (DHP) synthesized from coniferyl alcohol. The images obtained elucidate this structure for the first time. The structure of DHP, as seen from STM images, shows long-range order. It appears that DHP consists of building units or modules assembled into larger assemblies called supermodules. Supermodules are interconnected into the overall lattice-like polymer structure with or without spherical regions. One module consists of about 20 monomers, while the supermodule contains about 500 monomers. Calculated molecular weights for modules and supermodules agree with DHP molecular weight distribution peaks. Samples prepared at two different pH values, 6.4 and 7.6, have the same characteristics. The results presented demonstrate that the process of lignification, even in in vitro conditions, is highly ordered, and as such contribute to our understanding of the structure of lignin, a significant constitutive and functional element of cell walls.     

A scanning electron microscope study of a microsporidian with a pansporoblast: Thelohania maenadis

Air drying is not adequate for the preservation of the pansporoblastic membrane of Thelohania maenadis (Protozoa, Microsporidia), a parasite of the crabs Carcinus mediterraneus and Carcinus maenas. Freeze-drying and critical point drying preserve the pansporoblast membrane and reveal that isolated spores of the microsporidian are covered with a thick hairy coat. This coat originates as secretory product within the pansporoblast cavity.