Atomic force microscopy of single- and double-stranded DNA.

A method has been developed for imaging single-stranded DNA with the atomic force microscope (AFM). phi X174 single-stranded DNA in formaldehyde on mica can be imaged in the AFM under propanol or butanol or in air. Measured lengths of most molecules are on the order of 1 mu, although occasionally more extended molecules with lengths of 1.7 to 1.9 mu are seen. Single-stranded DNA in the AFM generally appears lumpier than double-stranded DNA, even when extended. Images of double-stranded lambda DNA in the AFM show more sharp kinks and bends than are typically observed in the electron microscope. Dense, aggregated fields of double-stranded plasmids can be converted by gentle rinsing with hot water to well spread fields.     

Applications for atomic force microscopy of DNA.

Tapping mode atomic force microscopy (AFM) of DNA in propanol, dry helium, and aqueous buffer each have specific applications. Resolution is best in propanol, which precipitates and immobilizes the DNA and provides a fluid imaging environment where adhesive forces are minimized. Resolution on exceptional images of DNA appears to be approximately 2 nm, sufficient to see helix turns in detail, but the smallest substructures typically seen on DNA in propanol are approximately 6-10 nm in size. Tapping AFM in dry helium provides a convenient way of imaging such things as conformations of DNA molecules and positions of proteins on DNA. Images of single-stranded DNA and RecA-DNA complexes are presented. In aqueous buffer DNA molecules as small as 300 bp have been imaged even when in motion. Images are presented of the changes in shape and position of circular plasmid DNA molecules.     

Phase imaging of moving DNA molecules and DNA molecules replicated in the atomic force microscope.

Phase imaging with a tapping mode atomic force microscope (AFM) has many advantages for imaging moving DNA and DNA-enzyme complexes in aqueous buffers at molecular resolution. In phase images molecules can be resolved at higher scan rates and lower forces than in height images from the AFM. Higher scan rates make it possible to image faster processes. At lower forces the molecules are imaged more gently. Moving DNA molecules are also resolved more clearly in phase images than in height images. Phase images in tapping mode AFM show the phase difference between oscillation of the piezoelectric crystal that drives the cantilever and oscillation of the cantilever as it interacts with the sample surface. Phase images presented here show moving DNA molecules that have been replicated with Sequenase in the AFM and DNA molecules tethered in complexes with Escherichia coli RNA polymerase.     

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.     

FM-AFM constant height imaging and force curves: high resolution study of DNA-tip interactions

Interaction of the atomic force microscopy (AFM) tip with the sample can be invasive for soft samples. Frequency Modulation (FM) AFM is gentler because it allows scanning in the non‐contact regime where only attractive forces exist between the tip and the sample, and there is no sample compression. Recently, FM‐AFM was used to resolve the atomic structure of single molecules of pentacene and of carbon nanotubes. We are testing similar FM‐AFM‐based approaches to study biological samples. We present FM‐AFM experiments on dsDNA deposited on 3‐aminopropyltriethoxysilane modified mica in ultra high vacuum. With flexible samples such as DNA, the substrate flatness is a sub‐molecular resolution limiting factor. Non‐contact topographic images of DNA show variations that have the periodicity of the right handed helix of B‐form DNA – this is an unexpected result as dehydrated DNA is thought to assume the A‐form structure. Frequency shift maps at constant height allow working in the non‐monotonic frequency shift range, show a rich contrast that changes significantly with the tip‐sample separation, and show 0.2 to 0.4 nm size details on DNA. Frequency shift versus distance curves acquired on DNA molecules and converted in force curves show that for small molecules (height < 2.5 nm), there is a contribution to the interaction force from the substrate when the tip is on top of the molecules. Our data shine a new light on dehydrated and adsorbed DNA behavior. They show a longer tip‐sample interaction distance. These experiments may have an impact on nanotechnological DNA applications in non‐physiological environments such as DNA based nanoelectronics and nanotemplating. Copyright © 2012 John Wiley & Sons, Ltd.     

Electrostatically balanced subnanometer imaging of biological specimens by atomic force microscope.

To achieve high-resolution topographs of native biological macromolecules in aqueous solution with the atomic force microscope (AFM) interactions between AFM tip and sample need to be considered. Short-range forces produce the submolecular information of high-resolution topographs. In contrast, no significant high-resolution information is provided by the long-range electrostatic double-layer force. However, this force can be adjusted by pH and electrolytes to distribute the force applied to the AFM tip over a large sample area. As demonstrated on fragile biological samples, adjustment of the electrolyte solution results in a local reduction of both vertical and lateral forces between the AFM tip and proteinous substructures. Under such electrostatically balanced conditions, the deformation of the native protein is minimized and the sample surface can be reproducibly contoured at a lateral resolution of 0.6 nm.     

Atomic force microscopic study on topological structures of pBR322 DNA

Plasmid pBR322 DNA (0.5mg/mL) isolated from Escherichia coli HB101 was suspended in Tris-HCl-EDTA (1 mol/L - 0.1 mol/L, pH8.5); then a drop of the above solution was deposited on freshly cleaved mica substrate. After adsorption for about 1 min, the sample was stained with phosphotungstic acid. The residua] solution was removed with a piece of filter paper. Afterwards the sample was imaged with a home-made atomic force microscope (AFM) in air. The AFM images of pBR322 DNA with a molecular resolution have been obtained. These images show that pBR322 DNA exists in several different topological structures: (i) relaxed circular DNA with a different diameter; (ii) supercondensed DNA with different particle sizes; (iii) dimeric catenane connected by one relaxed circular molecule and another dose-compacted molecule which might be either supercoiled or intramolecular knotted form; (iv) oligomeric catenane with multiple irregular molecules in which DNA is interlocked into a complex oligomer; (v) possibly-existing     

A method for anchoring round shaped cells for atomic force microscope imaging.

More and more researchers are interested in imaging living (Henderson, 1994) or fixed cells in their natural environment using the atomic force microscope (AFM). However, the AFM tip interacts strongly with the sample, and its z range freedom is limited to a few micrometers. This means that the cells to be imaged have to be strongly attached to the substrate, and imaging is restricted to cells having a flattened shape. Here we propose a simple and inexpensive solution to overcome these limitations. The method we propose is trapping living round shaped cells in a Millipore filter with a pore size comparable to the dimensions of the cell. The highest part of some of the blocked cells protrude through the holes of the filter and can this way be easily observed using the AFM without detachment.     

Sensing specific molecular interactions with the atomic force microscope

One of the unique features of the atomic force microscope (AFM) is its capacity to measure interactions between tip and sample with high sensitivity and unparalleled spatial resolution. Since the development of methods for the functionalization of the tips, the versatility of the AFM has been expanded to experiments where specific molecular interactions are measured. For illustration, we present measurements of the interaction between complementary strands of DNA. A necessary prerequisite for the quantitative analysis of the interaction force is knowledge of the spring constant of the cantilevers. Here, we compare different techniques that allow for the in situ measurement of the absolute value of the spring constant of cantilevers.     

DNA binding to mica correlates with cationic radius: assay by atomic force microscopy.

In buffers containing selected transition metal salts, DNA binds to mica tightly enough to be directly imaged in the buffer in the atomic force microscope (AFM, also known as scanning force microscope). The binding of DNA to mica, as measured by AFM-imaging, is correlated with the radius of the transition metal cation. The transition metal cations that effectively bind DNA to mica are Ni(II), Co(II), and Zn(II), which have ionic radii from 0.69 to 0.74 A. In Mn(II), ionic radius 0.82 A, DNA binds weakly to mica. In Cd(II) and Hg(II), respective ionic radii of 0.97 and 1.1 A, DNA does not bind to mica well enough to be imaged with the AFM. These results may to relate to how large a cation can fit into the cavities above the recessed hydroxyl groups in the mica lattice, although hypotheses based on hydrated ionic radii cannot be ruled out. The dependence of DNA binding on the concentrations of the cations Ni(II), Co(II), or Zn(II) shows maximal DNA binding at approximately 1-mM cation. Mg(II) does not bind DNA tightly enough to mica for AFM imaging. Mg(II) is a Group 2 cation with an ionic radius similar to that of Ni(II). Ni(II), Co(II), and Zn(II) have anomalously high enthalpies of hydration that may relate to their ability to bind DNA to mica. This AFM assay for DNA binding to mica has potential applications for assaying the binding of other polymers to mica and other flat surfaces.     

Atomic force microscope investigation of large-circle DNA molecules

A circular bacterial artificial chromosome of 148.9kbp on human chromosome 3 has been extended and fixed on bare mica substrates using a developed fluid capillary flow method in evaporating liquid drops. Extended circular DNA molecules were imaged with an atomic force microscope (AFM) under ambient conditions. The measured total lengths of the whole DNA molecules were in agreement with sequencing analysis data with an error range of +/-3.6%. This work is important groundwork for probing single nucleotide polymorphisms in the human genome, mapping genomic DNA, manipulating biomolecular nanotechnology, and studying the interaction of DNA-protein complexes investigated by AFM.     

Atomic force microscopy of oriented linear DNA molecules labeled with 5nm gold spheres.

The atomic force microscope (AFM;1) can image DNA and RNA in air and under solutions at resolution comparable to that obtained by electron microscopy (EM) (2-7). We have developed a method for depositing and imaging linear DNA molecules to which 5nm gold spheres have been attached. The gold spheres facilitate orientation of the DNA molecules on the mica surface to which they are absorbed and are potentially useful as internal height standards and as high resolution gene or sequence specific tags. We show that by modulating their adhesion to the mica surface, the gold spheres can be moved with some degree of control with the scanning tip.     

Atomic force microscopy of pollen grains,cellulose microfibrils,and protoplasts

Summary Atomic force microscopy (AFM) holds unique prospects for biological microscopy, such as nanometer resolution and the possibility of measuring samples in (physiological) solutions. This article reports the results of an examination of various types of plant material with the AFM. AFM images of the surface of pollen grains ofKalanchoe blossfeldiana andZea mays were compared with field emission scanning electron microscope (FESEM) images. AFM reached the same resolutions as FESEM but did not provide an overall view of the pollen grains. Using AFM in torsion mode, however, it was possible to reveal differences in friction forces of the surface of the pollen grains. Cellulose microfibrils in the cell wall of root hairs ofRaphanus sativus andZ. mays were imaged using AFM and transmission electron microscopy (TEM). Imaging was performed on specimens from which the wall matrix had been extracted. The cell wall texture of the root hairs was depicted clearly with AFM and was similar to the texture known from TEM. It was not possible to resolve substructures in a single microfibril. Because the scanning tip damaged the fragile cells, it was not possible to obtain images of living protoplasts ofZ. mays, but images of fixed and dried protoplasts are shown. We demonstrate that AFM of plant cells reaches resolutions as obtained with FESEM and TEM, but obstacles still have to be overcome before imaging of living protoplasts in physiological conditions can be realized.Abbreviations AFM atomic force microscope - FESEM field emission scanning electron microscope - PyMS pyrolysis mass spectrometry - TEM transmission electron microscope     

Atomic force microscopy of the myosin molecule.

Atomic force microscopy (AFM) has been used to study the structure of rabbit skeletal muscle myosin deposited onto a mica substrate from glycerol solution. Images of the myosin molecule have been obtained using contact mode AFM with the sample immersed in propanol. The molecules have two heads at one end of a long tail and have an appearance similar to those prepared by glycerol deposition techniques for electron microscopy, except that the separation of the two heads is not so well defined. The average length of the tail (155 +/- 5 nm) agrees well with previous studies. Bends in the myosin tail have been observed at locations similar to those observed in the electron microscope. By raising the applied force, it has been possible locally to separate the two strands of the alpha-helical coiled-coil tail. We conclude that the glycerol-mica technique is a useful tool for the preparation of fibrous proteins for examination by scanning probe microscopy.     

原子力显微镜拉伸吸附在金膜表面的短单链DNA分子

对单根DNA分子的操纵和拉伸可以直接研究DNA的弹性等力学性质. 首先通过将金沉积到云母表面制备了表面粗糙度小于0.3 nm的金膜,然后一段硫代的单链DNA (100 bases) 吸附到金膜表面. 利用原子力显微镜观察不同浓度的DNA吸附在金膜上的表面形貌. 进一步用原子力显微镜的力曲线模式拉伸DNA分子,在50％的情况下DNA可以被针尖拉伸,观察到了由于针尖和DNA分子间作用力的不同导致的多种不同力曲线.     

Photodynamic effect on melanoma cells investigated by atomic force microscopy

Atomic force microscopy (AFM) is a modern experimental method for imaging of conducting or non-conducting samples. New trends in the application of scanning probe microscopy (SPM) give us the ability to scan live cells directly in their ingenuous surroundings or in air. Our apparatus was replenished with an inverse optical microscope, so we could observe the position of the scanning tip in every individual cell. The aim of the presented study is to picture the cell surface in air. A dry scanner in non-contact or tapping mode was used in the biological application of AFM. In our work the cell line G361 was used as a biological sample. We imaged the cell line before and after induction of a photodynamic effect (PDE) by irradiation of ZnTPPS4-loaded cells with a light dose of 15 J/cm(2). Individual cells before PDE induction had a smooth surface without protrusion on the entire surface. Cells after PDE induction did not have a smooth surface but their surface was rough with protrusion and in some places cleaved.     

Atomic force microscopy of long and short double-stranded, single-stranded and triple-stranded nucleic acids.

Atomic force microscopy (AFM, also called scanning force microscopy) is proving to be a useful technique for imaging DNA. Thus it is important to push the limits of AFM imaging in order to explore both what types of DNA can be reliably imaged and identified and also what substrates and methods of sample preparation are suitable. The following advances in AFM of DNA are presented here. (i) DNA molecules as short as 25 bases can be seen by AFM. The short single-stranded DNAs imaged here (25 and 50 bases long) appeared globular in the AFM, perhaps because they are all capable of intramolecular base pairing and because the DNAs were in a Mg(ll) buffer, which facilitates intramolecular cross-bridging. (ii) AFM images in air of short double-stranded DNA molecules, 100-200 bp, gave lengths consistent with A-DNA. (iii) AFM images of poly (A) show both short bent lumpy molecules with an apparent persistence length of 40 nm and long straight molecules with an apparent persistence length of 600 nm. For comparison, the apparent persistence length for double-stranded DNA from phX-174 under the same conditions was 80 nm. (iv) Structures believed to be triple- stranded DNA were seen in samples of poly(dA.poly(dT) and poly (dG).poly(dC). These structures were twice as high as double-stranded DNA and the same width. (v) Entire molecules of lambda DNA, approx. 16 micron long, were imaged clearly in overlapping scans. (vi) Plasmid DNA was imaged on oxidized silicon, although less clearly than on mica.     

The intrinsic resolution limit in the atomic force microscope: implications for heights of nano-scale features

Background

Accurate mechanical characterization by the atomic force microscope at the highest spatial resolution requires that topography is deconvoluted from indentation. The measured height of nanoscale features in the atomic force microscope (AFM) is almost always smaller than the true value, which is often explained away as sample deformation, the formation of salt deposits and/or dehydration. We show that the real height of nano-objects cannot be obtained directly: a result arising as a consequence of the local probe-sample geometry.

Methods and Findings

We have modeled the tip-surface-sample interaction as the sum of the interaction between the tip and the surface and the tip and the sample. We find that the dynamics of the AFM cannot differentiate between differences in force resulting from 1) the chemical and/or mechanical characteristics of the surface or 2) a step in topography due to the size of the sample; once the size of a feature becomes smaller than the effective area of interaction between the AFM tip and sample, the measured height is compromised. This general result is a major contributor to loss of height and can amount to up to ∼90% for nanoscale features. In particular, these very large values in height loss may occur even when there is no sample deformation, and, more generally, height loss does not correlate with sample deformation. DNA and IgG antibodies have been used as model samples where experimental height measurements are shown to closely match the predicted phenomena.

Conclusions

Being able to measure the true height of single nanoscale features is paramount in many nanotechnology applications since phenomena and properties in the nanoscale critically depend on dimensions. Our approach allows accurate predictions for the true height of nanoscale objects and will lead to reliable mechanical characterization at the highest spatial resolution.     

Fabrication of a new substrate for atomic force microscopic observation of DNA molecules from an ultrasmooth sapphire plate.

A new stable substrate applicable to the observation of DNA molecules by atomic force microscopy (AFM) was fabricated from a ultrasmooth sapphire (alpha-Al2O3 single crystal) plate. The atomically ultrasmooth sapphire as obtained by high-temperature annealing has hydrophobic surfaces and could not be used for the AFM observation of DNA. However, sapphire treated with Na3PO4 aqueous solution exhibited a hydrophilic character while maintaining a smooth surface structure. The surface of the wet-treated sapphire was found by x-ray photoelectron spectroscopy and AFM to be approximately 0.3 nm. The hydrophilic surface character of the ultrasmooth sapphire plate made it easy for DNA molecules to adhere to the plate. Circular molecules of the plasmid DNA could be imaged by AFM on the hydrophilic ultrasmooth sapphire plate.